SloinpersName, BikovskipersName, LevipersName, Amber-VitospersName, KatzpersName, SpivakpersName, SomeckpersName, GattegnopersName (2022date). “Hybrid Offspring of C57BL/6J Mice Exhibit Improved Properties for Neurobehavioral Research.” eNeurotitle·j.
Object of study — whether first-generation offspring of two inbred strainsterm are a preferable model for neurobehavioral studies
Research question — how to balanceterm convenient geneticterm control against the complex behaviorterm that inbred strainsterm cannot support
Contribution — a single well-defined outbreeding step produces larger offspring that exhibit enhanced behaviorterm even during electrophysiology experiments, maintaining the ability of geneticterm targeting
phenomenon · claim-type · cross-domain-transfer · mechanism · claim-relation · replication-status · limitation — grounded in the full text| phenomenon | hybrid vigour in laboratory mouse phenotype — “In line with the established principle of hybrid vigor, we found that first-generation offspring of C57BL/6J and FVB/NJ mice exhibit improved learning and memory, maintain sensorimotor performance on aging, are larger, and exhibit improved behavior while carrying a chronic implant.” | |
| claim-type | method — “The hybrid mice provide an improved model for neurobehavioral studies combining complex behavior, electrophysiology, and genetic tools readily available in C57BL/6 mice.” | |
| method — evidence | — “Animals assigned to Battery A were subjected to assays in the following order: elevated plus maze (1 d); open field (1 d); rotarod (5 d); treadmill (3 d); optomotor drum (1 d); and forced swim test (1 d).” | |
| object of study — evidence | — “We bred FVB/NJ females and C57BL/6J males to generate first-generation hybrid offspring (FVB/NJ x C57BL/6J)F1. The hybrid mice exhibit reduced anxiety-like behavior, improved learning, and enhanced long-term spatial memory. In contrast to both progenitors, hybrids maintain sensorimotor performance upon aging and exhibit improved long-term memory.” | |
| research question — evidence | — “Because of genetic tools, mice are increasingly used for neuroscience experiments that were traditionally performed using rats. However, mice are smaller and “less intelligent” than rats, limiting the size of neural implants and the complexity of performed behaviors. Here, we show that these problems are exacerbated by the widespread use of inbred C57BL/6J mice, which are small animals with behavioral and sensorimotor deficits that worsen with age. In line with the established principle of hybrid vigor, we found that first-generation offspring of C57BL/6J and FVB/NJ mice exhibit improved learning and memory, maintain sensorimotor performance on aging, are larger, and exhibit improved behavior while carrying a chronic implant. The hybrid mice allow genetic control while supporting complex behavior over prolonged durations.” | |
| contribution — evidence | — “A single well-defined outbreeding step produces larger offspring that exhibit enhanced behavior even during electrophysiology experiments, maintaining the ability of genetic targeting.” | |
| transfer | from classical genetics: hybrid vigor, applied so that the first-generation offspring keep one transgenic allele from the C57 parent while losing the recessive traits of both — “Because of genetic tools, mice are increasingly used for neuroscience experiments that were traditionally performed using rats. However, mice are smaller and “less intelligent” than rats, limiting the size of neural implants and the complexity of performed behaviors. Here, we show that these problems are exacerbated by the widespread use of inbred C57BL/6J mice, which are small animals with behavioral and sensorimotor deficits that worsen with age. In line with the established principle of hybrid vigor, we found that first-generation offspring of C57BL/6J and FVB/NJ mice exhibit improved learning and memory, maintain sensorimotor performance on aging, are larger, and exhibit improved behavior while carrying a chronic implant. The hybrid mice allow genetic control while supporting complex behavior over prolonged durations.” | transfers[0] |
| transfer | from clinical phenotyping: the comparison with rats, where MICE ARE SMALLER AND LESS INTELLIGENT, LIMITING THE SIZE OF NEURAL IMPLANTS AND THE COMPLEXITY OF PERFORMED BEHAVIORS — “Because of genetic tools, mice are increasingly used for neuroscience experiments that were traditionally performed using rats. However, mice are smaller and “less intelligent” than rats, limiting the size of neural implants and the complexity of performed behaviors. Here, we show that these problems are exacerbated by the widespread use of inbred C57BL/6J mice, which are small animals with behavioral and sensorimotor deficits that worsen with age. In line with the established principle of hybrid vigor, we found that first-generation offspring of C57BL/6J and FVB/NJ mice exhibit improved learning and memory, maintain sensorimotor performance on aging, are larger, and exhibit improved behavior while carrying a chronic implant. The hybrid mice allow genetic control while supporting complex behavior over prolonged durations.” | transfers[1] |
| transfer | from animal welfare: the 3Rs argument, derived here from the behavioral result rather than asserted separately — “The hybrid vigor and the enhanced behavioral capabilities may yield more trials in every experimental session, shorter learning periods, and higher accuracy in complex tasks. By increasing yield and accuracy, the usage of HYB may allow uncovering unexplored neuronal mechanisms. Enhanced behavior also has ethical advantages, since faster learning and higher yield per animal translate to fewer experimental animals or sessions required to answer a given question.” | transfers[2] |
| mechanism | Hybrid vigor: crossing two inbred strains of distinct genealogy restores heterozygosity in the F1, which yields larger, better-learning, slower-declining animals — “In line with the established principle of hybrid vigor, we found that first-generation offspring of C57BL/6J and FVB/NJ mice exhibit improved learning and memory, maintain sensorimotor performance on aging, are larger, and exhibit improved behavior while carrying a chronic implant.” | mechanisms[0] |
| mechanism | Inbreeding drives the deficits it is meant to control: homozygosity exposes recessive alleles and genetic drift destabilises the strain across generations — “However, inbreeding exposes undesired phenotypical traits because of homozygous recessive alleles and does not guarantee stability over generations because of genetic drift ( Brekke et al., 2018 ).” | mechanisms[1] |
| mechanism | The hybrids' poorer rotarod balance is attributed to their greater body weight rather than to a motor deficit, because weight itself degrades rotarod performance — “HYB impaired balance may result from weight differences between the strains since HYB weigh more, and weight affects rotarod performance ( Mao et al., 2015 ).” | mechanisms[2] |
| mechanism | Light spreads further in neocortex than in CA1 because recurrent excitatory connectivity lets directly activated cells recruit distant ones, a connectivity CA1 lacks — “The difference between brain regions is consistent with different network topologies, since in contrast to the neocortex, CA1 PYR do not exhibit abundant recurrent excitation ( Thomson and Radpour, 1991 ; Buhl and Whittington, 2007 ).” | mechanisms[3] |
| mechanism | Implant weight is capped by body size, so a larger animal permits heavier headgear and hence more complex behavioral experiments — “Since headgear weight is limited by body size, the range of studied behaviors is limited by animal weight.” | mechanisms[4] |
| mechanism | inbreeding EXPOSES UNDESIRED PHENOTYPICAL TRAITS because of homozygous recessive alleles, and DOES NOT GUARANTEE STABILITY OVER GENERATIONS because of genetic drift — “However, inbreeding exposes undesired phenotypical traits because of homozygous recessive alleles and does not guarantee stability over generations because of genetic drift ( Brekke et al., 2018 ).” | mechanisms[5] |
| mechanism | and the hybrid inherits one allele from each parent, so the recessive traits are not expressed - 25% of the F2 generation would be blind because of a functional copy of the Pde6b gene MAINTAINED IN EVERY F1 HYBRID — “We focused on first generation (F1) hybrids to minimize genetic differences between individuals. While further breeding is expected to increase intersubject genetic variability, F2 hybrids will necessarily express recessive traits not expressed by the F1 generation. For instance, 25% of the F2 hybrids are expected to be blind because of a functional copy of the Pde6b gene ( Pittler and Baehr, 1991 ) maintained in every F1 hybrid. Thus, unless careful backcrossing is performed, F2 hybrids should not be used.” | mechanisms[6] |
| mechanism | SINCE HEADGEAR WEIGHT IS LIMITED BY BODY SIZE, THE RANGE OF STUDIED BEHAVIORS IS LIMITED BY ANIMAL WEIGHT - and the hybrids are heavier than either progenitor — “C57 mice are also smaller than other strains, which is a disadvantage for electrophysiological studies involving implanted headgear. Since headgear weight is limited by body size, the range of studied behaviors is limited by animal weight.” | mechanisms[7] |
| builds-on | Blednov et al. (2005, 2010), on C57BL/6J x FVB/NJ offspring as an alcohol-consumption model — “Previous work has shown that offspring of C57BL/6J (C57) and FVB/NJ (FVB) mice consume more alcohol than either progenitor, serving as a preferable model for alcohol consumption ( Blednov et al., 2005 , 2010 ).” | claim_relations[0] |
| replicates | Stark et al. (2012), distance-dependent optogenetic activation in rat hippocampus — “As previously reported in rat hippocampus ( Stark et al., 2012 ), activation probability depended on the horizontal distance from the light source.” | claim_relations[1] |
| replicates | Stark et al. (2013), indirect interneuron activation in C57 mice — “Thus, consistent with previous observations in C57 mice ( Stark et al., 2013 ), indirect INT activation in CA1 leads to more widespread activation than the local activation achieved in PYR.” | claim_relations[2] |
| critiques | the widespread use of one inbred strain, whose deficits limit which behavioral tasks can be studied at all — “C57BL/6-derived mice exhibit known phenotypic disadvantages, including sensitivity to pain, addiction, impaired balance, age-dependent hearing loss, and increased anxiety ( Crawley, 1996 ; Mogil et al., 1999 ; Ouagazzal et al., 2006 ). Thus, the behavioral tasks studied using C57BL/6 are limited.” | claim_relations[4] |
| extends | the established principle of hybrid vigor, applied to the specific pairing that keeps C57-background transgenic tools usable — “Because of genetic tools, mice are increasingly used for neuroscience experiments that were traditionally performed using rats. However, mice are smaller and “less intelligent” than rats, limiting the size of neural implants and the complexity of performed behaviors. Here, we show that these problems are exacerbated by the widespread use of inbred C57BL/6J mice, which are small animals with behavioral and sensorimotor deficits that worsen with age. In line with the established principle of hybrid vigor, we found that first-generation offspring of C57BL/6J and FVB/NJ mice exhibit improved learning and memory, maintain sensorimotor performance on aging, are larger, and exhibit improved behavior while carrying a chronic implant. The hybrid mice allow genetic control while supporting complex behavior over prolonged durations.” | claim_relations[5] |
| supports | strain selection as a design decision that can affect research outcome, because strain characteristics interact with the manipulations under test — “One of the challenges in mouse-based research is adjusting the strain to the specific study question. Strain selection can potentially affect research outcome, since strain characteristics may interact with genetic manipulations ( Sittig et al., 2016 ). Furthermore, strain characteristics could interact with experimental manipulations. For example, mice that suffer from retinal degeneration cannot fully use visual information and exhibit impaired behavior in hippocampal-based navigation tasks that rely on visual cues” | claim_relations[6] |
| predicts | that female hybrids and the reciprocal cross may behave differently, and that both need full characterisation before use — “Second, we employed HYB which are offspring of FVB females and C57 males. Full characterization is required before the use of reciprocal hybrids, offspring of a C57 female and an FVB male. However, because of the reduced litter sizes of female C57 compared with female FVB ( Taketo et al., 1991 ; Silver, 1995 ), the usage of the reciprocal hybrids is not recommended. Finally, since interstrain differences are also sex-dependent ( Brown and Wong, 2007 ), female HYB may exhibit distinct phenotypes.” | claim_relations[7] |
| sense | TWO WAYS OF SCORING ONE TASK GIVE OPPOSITE ANSWERS: when performance is quantified by the FRACTION OF TIME SPENT in the target quadrant, THERE IS NO CONSISTENT DIFFERENCE BETWEEN THE STRAINS - when it is quantified by the FRACTION OF VISITS to the same quadrant, HYB EXHIBIT IMPROVED PERFORMANCE. Same animals, same trials, same figure — “Spatial long-term memory behavior on the MWM. a , When performance is quantified by the fraction of time spent in the target quadrant, there is no consistent difference between the strains. b , When performance is quantified by the fraction of visits to the target quadrant, HYB exhibit improved performance compared with C57 and to FVB.” | senses[7] |
| sense | THE MODEL DECIDES WHAT CAN BE STUDIED: SINCE HEADGEAR WEIGHT IS LIMITED BY BODY SIZE, THE RANGE OF STUDIED BEHAVIORS IS LIMITED BY ANIMAL WEIGHT — “C57 mice are also smaller than other strains, which is a disadvantage for electrophysiological studies involving implanted headgear. Since headgear weight is limited by body size, the range of studied behaviors is limited by animal weight.” | senses[8] |
| sense | and the same point about the strain's deficits: C57BL/6-derived mice exhibit sensitivity to pain, addiction, impaired balance, age-dependent hearing loss and increased anxiety - THUS, THE BEHAVIORAL TASKS STUDIED USING C57BL/6 ARE LIMITED — “C57BL/6-derived mice exhibit known phenotypic disadvantages, including sensitivity to pain, addiction, impaired balance, age-dependent hearing loss, and increased anxiety ( Crawley, 1996 ; Mogil et al., 1999 ; Ouagazzal et al., 2006 ). Thus, the behavioral tasks studied using C57BL/6 are limited.” | senses[9] |
| sense | STRAIN SELECTION CAN POTENTIALLY AFFECT RESEARCH OUTCOME, since strain characteristics MAY INTERACT WITH GENETIC MANIPULATIONS and with experimental ones - mice with retinal degeneration CANNOT FULLY USE VISUAL INFORMATION and are impaired on navigation tasks that rely on visual cues — “One of the challenges in mouse-based research is adjusting the strain to the specific study question. Strain selection can potentially affect research outcome, since strain characteristics may interact with genetic manipulations ( Sittig et al., 2016 ). Furthermore, strain characteristics could interact with experimental manipulations. For example, mice that suffer from retinal degeneration cannot fully use visual information and exhibit impaired behavior in hippocampal-based navigation tasks that rely on visual cues” | senses[10] |
| sense | and the control group is named as a thing that can drift: A SECOND CHALLENGE IS TO ENSURE THE STABILITY OF THE TESTED PHENOTYPE UNDER THE NULL CONDITION - DETERIORATION OF CONTROL PHENOTYPIC BEHAVIOR COULD CONFOUND potential manipulation-based intergroup differences — “A second challenge is to ensure the stability of the tested phenotype under the null condition throughout the study period. In many cases, the effects of a given manipulation are studied over weeks to months ( Prevot et al., 2019 ; Namdar et al., 2020 ; Rahn et al., 2021 ). Deterioration of control phenotypic behavior could confound potential manipulation-based intergroup differences.” | senses[11] |
| sense | with inbreeding's own instability stated: it EXPOSES UNDESIRED PHENOTYPICAL TRAITS because of homozygous recessive alleles and DOES NOT GUARANTEE STABILITY OVER GENERATIONS BECAUSE OF GENETIC DRIFT — “However, inbreeding exposes undesired phenotypical traits because of homozygous recessive alleles and does not guarantee stability over generations because of genetic drift ( Brekke et al., 2018 ).” | senses[12] |
| sense | A STRAIN THAT CANNOT SEE, MEASURED RATHER THAN ASSUMED: FVB DID NOT MAKE ANY HEAD TURNS, CONSISTENT WITH BLINDNESS BECAUSE OF RETINAL DEGENERATION - one of the two progenitors of the proposed model is blind — “In contrast, FVB did not make any head turns (0 [0 0]; C57 vs FVB: p = 0.0011; HYB vs FVB: p = 0.003), consistent with blindness because of retinal degeneration ( Pittler and Baehr, 1991 ).” | senses[13] |
| sense | and the recessive trait is carried invisibly by the hybrid: 25% OF THE F2 HYBRIDS ARE EXPECTED TO BE BLIND because of a functional copy of the Pde6b gene MAINTAINED IN EVERY F1 HYBRID - so UNLESS CAREFUL BACKCROSSING IS PERFORMED, F2 HYBRIDS SHOULD NOT BE USED — “We focused on first generation (F1) hybrids to minimize genetic differences between individuals. While further breeding is expected to increase intersubject genetic variability, F2 hybrids will necessarily express recessive traits not expressed by the F1 generation. For instance, 25% of the F2 hybrids are expected to be blind because of a functional copy of the Pde6b gene ( Pittler and Baehr, 1991 ) maintained in every F1 hybrid. Thus, unless careful backcrossing is performed, F2 hybrids should not be used.” | senses[14] |
| sense | THE EXCEPTIONS ARE LISTED BY THE AUTHORS, EACH WITH THE USE IT DISQUALIFIES: similar or improved performance in every assay WITH ONE EXCEPTION, BALANCE at three months - so HYBRIDS MAY BE UNSUITABLE FOR STUDIES INVOLVING BALANCE — “There are some situations in which hybrid mice are not advantageous. First, compared with the C57 progenitors, HYB exhibited similar or improved performance in every assay used, with one exception: balance performance in the three-month-old age group. HYB impaired balance may result from weight differences between the strains since HYB weigh more, and weight affects rotarod performance ( Mao et al., 2015 ). Although HYB balance performance improved upon aging, hybrids may be unsuitable for studies involving balance.” | senses[15] |
| sense | and reduced anxiety cuts both ways - beneficial for cognitive and motor tasks, but the reduced anxiety and the age-related changes MAKE THE HYB LESS SUITABLE FOR LONG-TERM STUDIES OF ANXIETY — “Second, while the reduced anxiety exhibited by HYB is beneficial for studies involving cognitive and motor tasks, reduced anxiety-like behavior may be suboptimal for other purposes. For instance, the overall reduced anxiety-like behavior and the age-related changes make the HYB less suitable for long-term studies of anxiety.” | senses[16] |
| sense | AND THE MODEL'S OWN ROBUSTNESS IS NAMED AS A LIABILITY: BECAUSE HYB MICE ARE MORE ROBUST, GENETIC KNOCK-OUT MANIPULATIONS MAY BE LESS LIKELY TO SHOW A CONSISTENT PHENOTYPIC CHANGE - the healthier animal hides the effect being looked for — “Third, because HYB mice are more robust, genetic knock-out manipulations may be less likely to show a consistent phenotypic change ( Sittig et al., 2016 ). Fourth, it is not straightforward to use hybrids if a homozygous allele is required or if knock-out of both copies of a given gene is required (although see Silva et al., 1997 ).” | senses[17] |
| sense | with a dominant mutation carried by one progenitor: FVB CONTAIN A DISC1 MUTATION WHICH HAS BEEN SHOWN TO CAUSE COGNITIVE IMPAIRMENT EVEN WHEN HETEROZYGOUS - and the paper weighs it against inbreeding rather than dismissing it — “Third, some genes expressed by progenitors may be dominant; for instance, FVB contain a Disc1 mutation which has been shown to cause cognitive impairment even when heterozygous ( Koike et al., 2006 ; Ritchie and Clapcote, 2013 ). Nevertheless, we found that the potentially deleterious effects of dominant genes on HYB are smaller than the adverse effects of inbreeding.” | senses[18] |
| sense | and two whole conditions left uncharacterized: FULL CHARACTERIZATION IS REQUIRED BEFORE THE USE OF RECIPROCAL HYBRIDS, and FEMALE HYB MAY EXHIBIT DISTINCT PHENOTYPES - every number in this paper is from male mice of one cross direction — “Second, we employed HYB which are offspring of FVB females and C57 males. Full characterization is required before the use of reciprocal hybrids, offspring of a C57 female and an FVB male. However, because of the reduced litter sizes of female C57 compared with female FVB ( Taketo et al., 1991 ; Silver, 1995 ), the usage of the reciprocal hybrids is not recommended. Finally, since interstrain differences are also sex-dependent ( Brown and Wong, 2007 ), female HYB may exhibit distinct phenotypes.” | senses[19] |
| sense | the size result stated with its purpose attached: HYB were LARGER THAN EITHER PARENTAL STRAIN, and HIGHER WEIGHTS ARE BENEFICIAL FOR ELECTROPHYSIOLOGICAL EXPERIMENTS in freely-moving animals, IN WHICH ANIMAL SIZE LIMITS THE WEIGHT OF THE IMPLANTED APPARATUS — “We found that sedentary HYB were larger than either parental strain. At the age of three months, the median [IQR] weight of HYB was 32 [29.6 34.9] g, compared with 26.9 [25.1 28.1] g for C57 ( p = 9.6 × 10 −10 ; Kruskal–Wallis test, corrected for multiple comparisons; Fig. 1 ; Extended Data Fig. 1-1 A ). Higher weights are beneficial for electrophysiological experiments in freely-moving animals, in which animal size limits the weight of the implanted apparatus.” | senses[20] |
| sense | and the one assay where the hybrids do worse, reported plainly: HYB LATENCY TO FALL DID NOT INCREASE OVER DAYS - no balance learning, where both progenitors showed it — “HYB latency to fall did not increase over days ( p = 0.12, Kruskal–Wallis test). To summarize, at the age of three months, visual behavior, and motor endurance of HYB are similar to those of C57.” | senses[21] |
| sense | with an ethical argument made from a behavioral one: faster learning and higher yield per animal TRANSLATE TO FEWER EXPERIMENTAL ANIMALS OR SESSIONS REQUIRED TO ANSWER A GIVEN QUESTION — “The hybrid vigor and the enhanced behavioral capabilities may yield more trials in every experimental session, shorter learning periods, and higher accuracy in complex tasks. By increasing yield and accuracy, the usage of HYB may allow uncovering unexplored neuronal mechanisms. Enhanced behavior also has ethical advantages, since faster learning and higher yield per animal translate to fewer experimental animals or sessions required to answer a given question.” | senses[22] |
| control | the same three strains tested at two ages rather than one — held — rules out an advantage that exists only at the age tested - and it is what showed that the progenitors deteriorate while the hybrids do not — “Compared with the parental C57BL/6J progenitor strain, young first-generation hybrid offspring (FVB/NJ x C57BL/6J)F1 exhibited reduced anxiety-like behavior. Upon aging, HYB anxiety-like behavior was further reduced, and learning and long-term memory performance improved. In contrast, learning and memory performance of the parental C57BL/6J and the maternal FVB/NJ progenitor strains did not improve at older age. Furthermore, motor performance of the progenitor strains deteriorated upon aging, whereas HYB sensorimotor abilities were maintained. HYB were larger, ran faster, and performed more trials during electrophysiological experiments on the linear track. HYB and C57 exhibited similar CA1 place cell rate and phase coding. Finally, optogenetic manipulations were readily achieved in transgenic HYB.” | controls[0] |
| control | an optomotor drum test of visual behavior run before the cognitive assays — held — rules out reading a navigation deficit as cognitive when it is visual - FVB DID NOT MAKE ANY HEAD TURNS, CONSISTENT WITH BLINDNESS because of retinal degeneration — “In contrast, FVB did not make any head turns (0 [0 0]; C57 vs FVB: p = 0.0011; HYB vs FVB: p = 0.003), consistent with blindness because of retinal degeneration ( Pittler and Baehr, 1991 ).” | controls[1] |
| control | gait analysis and home cage behavior added to the standard battery — held — rules out a difference in the assays being read as a difference in the animals - the weight, daily conduct and gait differences together show the hybrid's spontaneous behavior IS DISTINCT FROM THAT OF BOTH PROGENITOR STRAINS — “Together, the weight, daily conduct, and gait differences indicate that the spontaneous behavior of the (FVB/NJ x C57BL/6J)F1 hybrids is distinct from that of both progenitor strains.” | controls[2] |
| control | long-term memory scored two different ways on the same task — partial — rules out nothing, and that is the point - by fraction of TIME in the target quadrant there is NO CONSISTENT DIFFERENCE between strains, by fraction of VISITS the hybrids are better — “Spatial long-term memory behavior on the MWM. a , When performance is quantified by the fraction of time spent in the target quadrant, there is no consistent difference between the strains. b , When performance is quantified by the fraction of visits to the target quadrant, HYB exhibit improved performance compared with C57 and to FVB.” | controls[3] |
| differs in | the widespread use of one inbred strain, whose deficits limit which behavioral tasks can be studied at all — THE AXIS IS THE STRAIN'S PHENOTYPE, AND IT CONSTRAINS WHICH QUESTIONS CAN BE ASKED. 'C57BL/6-derived mice exhibit known phenotypic disadvantages, including sensitivity to pain, addiction, impaired balance, age-dependent hearing loss, and increased anxiety. Thus, the behavioral tasks studied using C57BL/6 are l[imited].' Five named deficits in the standard animal, and the consequence is a restriction on the QUESTION SET rather than on any one result - so the literature's shape follows from the strain. — “C57BL/6-derived mice exhibit known phenotypic disadvantages, including sensitivity to pain, addiction, impaired balance, age-dependent hearing loss, and increased anxiety ( Crawley, 1996 ; Mogil et al., 1999 ; Ouagazzal et al., 2006 ). Thus, the behavioral tasks studied using C57BL/6 are limited.” — “C57BL/6-derived mice exhibit known phenotypic disadvantages, including sensitivity to pain, addiction, impaired balance, age-dependent hearing loss, and increased anxiety” | claim_relations[4] |
| note | FVB —contrasted-with→ C57 — A PROGENITOR THAT CANNOT SEE - FVB DID NOT MAKE ANY HEAD TURNS, CONSISTENT WITH BLINDNESS BECAUSE OF RETINAL DEGENERATION — “In contrast, FVB did not make any head turns (0 [0 0]; C57 vs FVB: p = 0.0011; HYB vs FVB: p = 0.003), consistent with blindness because of retinal degeneration ( Pittler and Baehr, 1991 ).” | concept_edges[8] |
| note | HYB —contrasted-with→ C57 — HYB SPENT MORE TIME IN THE OPEN ARMS THAN C57, INDICATING REDUCED ANXIETY-LIKE BEHAVIOR — “However, HYB spent more time in the open arms than C57, indicating reduced anxiety-like behavior ( p = 0.034; Kruskal–Wallis test; Fig. 2 A ; Extended Data Fig. 1-2 ).” | concept_edges[16] |
| outcome (stated by the tagger, no quotation) | control — held | controls[0] |
| outcome (stated by the tagger, no quotation) | control — held | controls[1] |
| outcome (stated by the tagger, no quotation) | control — held | controls[2] |
| outcome (stated by the tagger, no quotation) | control — partial | controls[3] |
| for which use | limitation — balance and motor studies specifically: ALTHOUGH HYB BALANCE PERFORMANCE IMPROVED UPON AGING, HYBRIDS MAY BE UNSUITABLE FOR STUDIES INVOLVING BALANCE — “Although HYB balance performance improved upon aging, hybrids may be unsuitable for studies involving balance.” | limitations[0] |
| for which use | limitation — long-term anxiety work: THE OVERALL REDUCED ANXIETY-LIKE BEHAVIOR AND THE AGE-RELATED CHANGES MAKE THE HYB LESS SUITABLE FOR LONG-TERM STUDIES OF ANXIETY — “For instance, the overall reduced anxiety-like behavior and the age-related changes make the HYB less suitable for long-term studies of anxiety.” | limitations[1] |
| for which use | limitation — the reciprocal cross, which was not characterised: FULL CHARACTERIZATION IS REQUIRED BEFORE THE USE OF RECIPROCAL HYBRIDS, OFFSPRING OF A C57 FEMALE AND AN FVB MALE — “Full characterization is required before the use of reciprocal hybrids, offspring of a C57 female and an FVB male.” | limitations[2] |
| for which use | limitation — designs needing homozygosity: IT IS NOT STRAIGHTFORWARD TO USE HYBRIDS IF A HOMOZYGOUS ALLELE IS REQUIRED OR IF KNOCK-OUT OF BOTH COPIES OF A GIVEN GENE IS REQUIRED — “Fourth, it is not straightforward to use hybrids if a homozygous allele is required or if knock-out of both copies of a given gene is required (although see Silva et al., 1997 ).” | limitations[3] |
| for which use | limitation — studies involving balance — “There are some situations in which hybrid mice are not advantageous. First, compared with the C57 progenitors, HYB exhibited similar or improved performance in every assay used, with one exception: balance performance in the three-month-old age group. HYB impaired balance may result from weight differences between the strains since HYB weigh more, and weight affects rotarod performance ( Mao et al., 2015 ). Although HYB balance performance improved upon aging, hybrids may be unsuitable for studies involving balance.” | limitations[4] |
| for which use | limitation — long-term studies of anxiety — “Second, while the reduced anxiety exhibited by HYB is beneficial for studies involving cognitive and motor tasks, reduced anxiety-like behavior may be suboptimal for other purposes. For instance, the overall reduced anxiety-like behavior and the age-related changes make the HYB less suitable for long-term studies of anxiety.” | limitations[5] |
| for which use | limitation — genetic knock-out manipulations, and any design requiring a homozygous allele — “Third, because HYB mice are more robust, genetic knock-out manipulations may be less likely to show a consistent phenotypic change ( Sittig et al., 2016 ). Fourth, it is not straightforward to use hybrids if a homozygous allele is required or if knock-out of both copies of a given gene is required (although see Silva et al., 1997 ).” | limitations[6] |
| for which use | limitation — any cognitive phenotype: SOME GENES EXPRESSED BY PROGENITORS MAY BE DOMINANT; FOR INSTANCE, FVB CONTAIN A Disc1 MUTATION WHICH HAS BEEN SHOWN TO CAUSE COGNITIVE IMPAIRMENT EVEN WHEN HETEROZYGOUS — “Third, some genes expressed by progenitors may be dominant; for instance, FVB contain a Disc1 mutation which has been shown to cause cognitive impairment even when heterozygous ( Koike et al., 2006 ; Ritchie and Clapcote, 2013 ). Nevertheless, we found that the potentially deleterious effects of dominant genes on HYB are smaller than the adverse effects of inbreeding.” | limitations[7] |
| for which use | limitation — the same scope as limitations[2], with the practical reason the reciprocal cross is rarely used: THE REDUCED LITTER SIZES OF FEMALE C57 COMPARED WITH FEMALE FVB — “Second, we employed HYB which are offspring of FVB females and C57 males. Full characterization is required before the use of reciprocal hybrids, offspring of a C57 female and an FVB male. However, because of the reduced litter sizes of female C57 compared with female FVB ( Taketo et al., 1991 ; Silver, 1995 ), the usage of the reciprocal hybrids is not recommended. Finally, since interstrain differences are also sex-dependent ( Brown and Wong, 2007 ), female HYB may exhibit distinct phenotypes.” | limitations[8] |
| for which use | limitation — any generation beyond the first: F2 HYBRIDS WILL NECESSARILY EXPRESS RECESSIVE TRAITS NOT EXPRESSED BY THE F1 GENERATION, so UNLESS CAREFUL BACKCROSSING IS PERFORMED, F2 HYBRIDS SHOULD NOT BE USED — “We focused on first generation (F1) hybrids to minimize genetic differences between individuals. While further breeding is expected to increase intersubject genetic variability, F2 hybrids will necessarily express recessive traits not expressed by the F1 generation. For instance, 25% of the F2 hybrids are expected to be blind because of a functional copy of the Pde6b gene ( Pittler and Baehr, 1991 ) maintained in every F1 hybrid. Thus, unless careful backcrossing is performed, F2 hybrids should not be used.” | limitations[9] |
| replication | untested | |
| limitation | Hybrids showed impaired balance at three months, so they may be unsuitable for balance studies. — “Although HYB balance performance improved upon aging, hybrids may be unsuitable for studies involving balance.” | limitations[0] |
| limitation | The hybrids' reduced and age-changing anxiety-like behavior makes them a poor model for long-term anxiety research. — “For instance, the overall reduced anxiety-like behavior and the age-related changes make the HYB less suitable for long-term studies of anxiety.” | limitations[1] |
| limitation | Only one cross direction (FVB dam x C57 sire) was characterized; the reciprocal cross was not tested. — “Full characterization is required before the use of reciprocal hybrids, offspring of a C57 female and an FVB male.” | limitations[2] |
| limitation | Hybrids are awkward for designs requiring homozygosity or double knock-out. — “Fourth, it is not straightforward to use hybrids if a homozygous allele is required or if knock-out of both copies of a given gene is required (although see Silva et al., 1997 ).” | limitations[3] |
| limitation | compared with the C57 progenitors, HYB exhibited similar or improved performance in every assay used, WITH ONE EXCEPTION: BALANCE PERFORMANCE in the three-month-old age group, so HYBRIDS MAY BE UNSUITABLE FOR STUDIES INVOLVING BALANCE — “There are some situations in which hybrid mice are not advantageous. First, compared with the C57 progenitors, HYB exhibited similar or improved performance in every assay used, with one exception: balance performance in the three-month-old age group. HYB impaired balance may result from weight differences between the strains since HYB weigh more, and weight affects rotarod performance ( Mao et al., 2015 ). Although HYB balance performance improved upon aging, hybrids may be unsuitable for studies involving balance.” | limitations[4] |
| limitation | REDUCED ANXIETY-LIKE BEHAVIOR MAY BE SUBOPTIMAL FOR OTHER PURPOSES - the overall reduced anxiety and the age-related changes make the HYB LESS SUITABLE FOR LONG-TERM STUDIES OF ANXIETY — “Second, while the reduced anxiety exhibited by HYB is beneficial for studies involving cognitive and motor tasks, reduced anxiety-like behavior may be suboptimal for other purposes. For instance, the overall reduced anxiety-like behavior and the age-related changes make the HYB less suitable for long-term studies of anxiety.” | limitations[5] |
| limitation | BECAUSE HYB MICE ARE MORE ROBUST, GENETIC KNOCK-OUT MANIPULATIONS MAY BE LESS LIKELY TO SHOW A CONSISTENT PHENOTYPIC CHANGE, and it is not straightforward to use hybrids if a homozygous allele is required — “Third, because HYB mice are more robust, genetic knock-out manipulations may be less likely to show a consistent phenotypic change ( Sittig et al., 2016 ). Fourth, it is not straightforward to use hybrids if a homozygous allele is required or if knock-out of both copies of a given gene is required (although see Silva et al., 1997 ).” | limitations[6] |
| limitation | some genes expressed by progenitors may be DOMINANT - FVB contain a Disc1 mutation shown to CAUSE COGNITIVE IMPAIRMENT EVEN WHEN HETEROZYGOUS — “Third, some genes expressed by progenitors may be dominant; for instance, FVB contain a Disc1 mutation which has been shown to cause cognitive impairment even when heterozygous ( Koike et al., 2006 ; Ritchie and Clapcote, 2013 ). Nevertheless, we found that the potentially deleterious effects of dominant genes on HYB are smaller than the adverse effects of inbreeding.” | limitations[7] |
| limitation | FULL CHARACTERIZATION IS REQUIRED BEFORE THE USE OF RECIPROCAL HYBRIDS, and since interstrain differences are also sex-dependent, FEMALE HYB MAY EXHIBIT DISTINCT PHENOTYPES — “Second, we employed HYB which are offspring of FVB females and C57 males. Full characterization is required before the use of reciprocal hybrids, offspring of a C57 female and an FVB male. However, because of the reduced litter sizes of female C57 compared with female FVB ( Taketo et al., 1991 ; Silver, 1995 ), the usage of the reciprocal hybrids is not recommended. Finally, since interstrain differences are also sex-dependent ( Brown and Wong, 2007 ), female HYB may exhibit distinct phenotypes.” | limitations[8] |
| limitation | UNLESS CAREFUL BACKCROSSING IS PERFORMED, F2 HYBRIDS SHOULD NOT BE USED — “We focused on first generation (F1) hybrids to minimize genetic differences between individuals. While further breeding is expected to increase intersubject genetic variability, F2 hybrids will necessarily express recessive traits not expressed by the F1 generation. For instance, 25% of the F2 hybrids are expected to be blind because of a functional copy of the Pde6b gene ( Pittler and Baehr, 1991 ) maintained in every F1 hybrid. Thus, unless careful backcrossing is performed, F2 hybrids should not be used.” | limitations[9] |
finding · quantity · variable · definition · key-term — grounded in the full text| finding | First-generation FVB/NJ x C57BL/6J hybrids are heavier than either parental strain. — “We found that sedentary HYB were larger than either parental strain.” | content_tags.findings[0] |
| finding | THE HYBRID MICE EXHIBIT REDUCED ANXIETY-LIKE BEHAVIOR, IMPROVED LEARNING, AND ENHANCED LONG-TERM SPATIAL MEMORY — “The hybrid mice exhibit reduced anxiety-like behavior, improved learning, and enhanced long-term spatial memory.” | content_tags.findings[1] |
| finding | At three months of age the hybrids already outperform both progenitor strains on Morris water maze learning. — “Hence, compared with both progenitors, three-month-old HYB exhibit improved learning performance.” | content_tags.findings[2] |
| finding | On the water maze probe day only the hybrids, not either progenitor strain, showed evidence of spatial long-term memory. — “Thus, in contrast to both progenitors, three-month-old HYB exhibit spatial long-term memory behavior.” | content_tags.findings[3] |
| finding | while the motor capabilities of C57 and FVB DETERIORATE UPON AGING, HYB MOTOR PERFORMANCE REMAINS STABLE across the two age groups studied — “Thus, while the motor capabilities of C57 and FVB deteriorate upon aging, HYB motor performance remains stable across the two age groups studied.” | content_tags.findings[4] |
| finding | anxiety-like behavior is REDUCED and memory performance IMPROVES ON AGING in HYB — “Thus, anxiety-like behavior is reduced and memory performance improves on aging in HYB” | content_tags.findings[5] |
| finding | During prolonged implanted recordings, freely-moving hybrids showed enhanced motor ability and exploration. — “Thus, freely-moving HYB exhibit enhanced motor abilities and exploration during prolonged electrophysiological recordings.” | content_tags.findings[6] |
| finding | CA1 pyramidal cells in hybrids and C57 mice show similar rate coding of space. — “Thus, CA1 PYR recorded in HYB and in C57 mice exhibit similar rate coding of space.” | content_tags.findings[7] |
| finding | Theta phase precession in CA1 place fields is also equivalent between hybrids and C57. — “Thus, CA1 PYR recorded in HYB and in C57 exhibit similar phase coding of space.” | content_tags.findings[8] |
| finding | Crossing transgenic C57 lines with FVB yields transgenic hybrid offspring suitable for optogenetic experiments. — “In summary, crossing transgenic C57 with FVB yields transgenic HYB offspring, suitable for optogenetic experiments.” | content_tags.findings[9] |
| finding | The single assay on which hybrids were worse than C57 was rotarod balance at three months of age. — “First, compared with the C57 progenitors, HYB exhibited similar or improved performance in every assay used, with one exception: balance performance in the three-month-old age group.” | content_tags.findings[10] |
| finding | FVB DO NOT REACT TO ROTATING VISUAL STIMULI AT EITHER AGE GROUP — “In contrast, FVB do not react to rotating visual stimuli at either age group.” | content_tags.findings[11] |
| finding | when performance is quantified by the FRACTION OF TIME SPENT IN THE TARGET QUADRANT, THERE IS NO CONSISTENT DIFFERENCE BETWEEN THE STRAINS; by the FRACTION OF VISITS to the target quadrant, HYB EXHIBIT IMPROVED PERFORMANCE — “Spatial long-term memory behavior on the MWM. a , When performance is quantified by the fraction of time spent in the target quadrant, there is no consistent difference between the strains. b , When performance is quantified by the fraction of visits to the target quadrant, HYB exhibit improved performance compared with C57 and to FVB.” | content_tags.findings[18] |
| finding | SINCE HEADGEAR WEIGHT IS LIMITED BY BODY SIZE, THE RANGE OF STUDIED BEHAVIORS IS LIMITED BY ANIMAL WEIGHT — “C57 mice are also smaller than other strains, which is a disadvantage for electrophysiological studies involving implanted headgear. Since headgear weight is limited by body size, the range of studied behaviors is limited by animal weight.” | content_tags.findings[19] |
| finding | THUS, THE BEHAVIORAL TASKS STUDIED USING C57BL/6 ARE LIMITED — “C57BL/6-derived mice exhibit known phenotypic disadvantages, including sensitivity to pain, addiction, impaired balance, age-dependent hearing loss, and increased anxiety ( Crawley, 1996 ; Mogil et al., 1999 ; Ouagazzal et al., 2006 ). Thus, the behavioral tasks studied using C57BL/6 are limited.” | content_tags.findings[20] |
| finding | STRAIN SELECTION CAN POTENTIALLY AFFECT RESEARCH OUTCOME, SINCE STRAIN CHARACTERISTICS MAY INTERACT WITH GENETIC MANIPULATIONS — “One of the challenges in mouse-based research is adjusting the strain to the specific study question. Strain selection can potentially affect research outcome, since strain characteristics may interact with genetic manipulations ( Sittig et al., 2016 ). Furthermore, strain characteristics could interact with experimental manipulations. For example, mice that suffer from retinal degeneration cannot fully use visual information and exhibit impaired behavior in hippocampal-based navigation tasks that rely on visual cues” | content_tags.findings[21] |
| finding | DETERIORATION OF CONTROL PHENOTYPIC BEHAVIOR COULD CONFOUND POTENTIAL MANIPULATION-BASED INTERGROUP DIFFERENCES — “A second challenge is to ensure the stability of the tested phenotype under the null condition throughout the study period. In many cases, the effects of a given manipulation are studied over weeks to months ( Prevot et al., 2019 ; Namdar et al., 2020 ; Rahn et al., 2021 ). Deterioration of control phenotypic behavior could confound potential manipulation-based intergroup differences.” | content_tags.findings[22] |
| finding | inbreeding EXPOSES UNDESIRED PHENOTYPICAL TRAITS because of homozygous recessive alleles and DOES NOT GUARANTEE STABILITY OVER GENERATIONS BECAUSE OF GENETIC DRIFT — “However, inbreeding exposes undesired phenotypical traits because of homozygous recessive alleles and does not guarantee stability over generations because of genetic drift ( Brekke et al., 2018 ).” | content_tags.findings[23] |
| finding | FVB DID NOT MAKE ANY HEAD TURNS, CONSISTENT WITH BLINDNESS BECAUSE OF RETINAL DEGENERATION — “In contrast, FVB did not make any head turns (0 [0 0]; C57 vs FVB: p = 0.0011; HYB vs FVB: p = 0.003), consistent with blindness because of retinal degeneration ( Pittler and Baehr, 1991 ).” | content_tags.findings[24] |
| finding | 25% OF THE F2 HYBRIDS ARE EXPECTED TO BE BLIND, so UNLESS CAREFUL BACKCROSSING IS PERFORMED, F2 HYBRIDS SHOULD NOT BE USED — “We focused on first generation (F1) hybrids to minimize genetic differences between individuals. While further breeding is expected to increase intersubject genetic variability, F2 hybrids will necessarily express recessive traits not expressed by the F1 generation. For instance, 25% of the F2 hybrids are expected to be blind because of a functional copy of the Pde6b gene ( Pittler and Baehr, 1991 ) maintained in every F1 hybrid. Thus, unless careful backcrossing is performed, F2 hybrids should not be used.” | content_tags.findings[25] |
| finding | HYBRIDS MAY BE UNSUITABLE FOR STUDIES INVOLVING BALANCE — “There are some situations in which hybrid mice are not advantageous. First, compared with the C57 progenitors, HYB exhibited similar or improved performance in every assay used, with one exception: balance performance in the three-month-old age group. HYB impaired balance may result from weight differences between the strains since HYB weigh more, and weight affects rotarod performance ( Mao et al., 2015 ). Although HYB balance performance improved upon aging, hybrids may be unsuitable for studies involving balance.” | content_tags.findings[26] |
| finding | the reduced anxiety-like behavior and the age-related changes MAKE THE HYB LESS SUITABLE FOR LONG-TERM STUDIES OF ANXIETY — “Second, while the reduced anxiety exhibited by HYB is beneficial for studies involving cognitive and motor tasks, reduced anxiety-like behavior may be suboptimal for other purposes. For instance, the overall reduced anxiety-like behavior and the age-related changes make the HYB less suitable for long-term studies of anxiety.” | content_tags.findings[27] |
| finding | BECAUSE HYB MICE ARE MORE ROBUST, GENETIC KNOCK-OUT MANIPULATIONS MAY BE LESS LIKELY TO SHOW A CONSISTENT PHENOTYPIC CHANGE — “Third, because HYB mice are more robust, genetic knock-out manipulations may be less likely to show a consistent phenotypic change ( Sittig et al., 2016 ). Fourth, it is not straightforward to use hybrids if a homozygous allele is required or if knock-out of both copies of a given gene is required (although see Silva et al., 1997 ).” | content_tags.findings[28] |
| finding | FVB CONTAIN A DISC1 MUTATION WHICH HAS BEEN SHOWN TO CAUSE COGNITIVE IMPAIRMENT EVEN WHEN HETEROZYGOUS — “Third, some genes expressed by progenitors may be dominant; for instance, FVB contain a Disc1 mutation which has been shown to cause cognitive impairment even when heterozygous ( Koike et al., 2006 ; Ritchie and Clapcote, 2013 ). Nevertheless, we found that the potentially deleterious effects of dominant genes on HYB are smaller than the adverse effects of inbreeding.” | content_tags.findings[29] |
| finding | FULL CHARACTERIZATION IS REQUIRED BEFORE THE USE OF RECIPROCAL HYBRIDS, and FEMALE HYB MAY EXHIBIT DISTINCT PHENOTYPES — “Second, we employed HYB which are offspring of FVB females and C57 males. Full characterization is required before the use of reciprocal hybrids, offspring of a C57 female and an FVB male. However, because of the reduced litter sizes of female C57 compared with female FVB ( Taketo et al., 1991 ; Silver, 1995 ), the usage of the reciprocal hybrids is not recommended. Finally, since interstrain differences are also sex-dependent ( Brown and Wong, 2007 ), female HYB may exhibit distinct phenotypes.” | content_tags.findings[30] |
| finding | HYB LATENCY TO FALL DID NOT INCREASE OVER DAYS — “HYB latency to fall did not increase over days ( p = 0.12, Kruskal–Wallis test). To summarize, at the age of three months, visual behavior, and motor endurance of HYB are similar to those of C57.” | content_tags.findings[31] |
| quantity | 176 — total number of freely-moving adult mice used in the study — “A total of 176 freely-moving adult mice were used in this study.” | content_tags.quantities[0] |
| quantity | 32 [29.6 34.9] g vs 26.9 [25.1 28.1] g — median [IQR] body weight of three-month-old hybrids versus C57 mice — “the median [IQR] weight of HYB was 32 [29.6 34.9] g, compared with 26.9 [25.1 28.1] g for C57” | content_tags.quantities[1] |
| quantity | 164 [139 215] vs 136 [113 178] — median [IQR] number of one-direction linear-track trials per session, hybrids versus C57 — “HYB conducted a median [IQR] of 164 [139 215] one-direction trials per session, while C57 ran 136 [113 178] trials” | content_tags.quantities[2] |
| quantity | 31 [22 44] cm/s vs 23 [18 30] cm/s — median [IQR] running speed on the linear track, hybrids versus C57 — “Furthermore, HYB exhibited higher running speeds, 31 [22 44] cm/s compared with 23 [18 30] cm/s for C57” | content_tags.quantities[3] |
| quantity | 0.36 [0.33 0.44] vs 0.28 [0.26 0.27] — fraction of visits to the target quadrant on the water-maze probe day, hybrids versus C57 (chance = 0.25) — “HYB exhibited the largest fraction of visits to the target quadrant, indicative of spatial long-term memory (C57: 0.28 [0.26 0.27]; FVB: 0.21 [0.15 0.3]; HYB: 0.36 [0.33 0.44]; p = 0.0014, Kruskal–Wallis test; Fig. 2 Cb ).” | content_tags.quantities[4] |
| quantity | 443/779 (57%) and 508/960 (53%) — place fields detected in 443/779 (57%) of C57 PYR and in 508/960 (53%) of HYB PYR — “Place fields were detected in 443/779 (57%) of C57 PYR and in 508/960 (53%) of HYB PYR ( Fig. 5 Eb ).” | content_tags.quantities[5] |
| quantity | 308/603 (51%) and 353/649 (54%) — of all fields, 308/603 (51%) C57 PYR and 353/649 (54%) HYB PYR exhibited CONSISTENT SPATIAL THETA PHASE PRECESSION — “Of all fields, 308/603 (51%) C57 PYR and 353/649 (54%) HYB PYR exhibited consistent spatial theta phase precession” | content_tags.quantities[6] |
| quantity | 772/1527 (47%) — proportion of CA1 pyramidal cells on the illuminated shank that were optogenetically activated in transgenic hybrids — “Of the PYR recorded on the illuminated shank, 772/1527 (47%) exhibited optical activation” | content_tags.quantities[7] |
| quantity | 25% — expected proportion of F2 hybrids that would be blind through homozygosity for the non-functional Pde6b allele — “For instance, 25% of the F2 hybrids are expected to be blind because of a functional copy of the Pde6b gene ( Pittler and Baehr, 1991 ) maintained in every F1 hybrid.” | content_tags.quantities[8] |
| quantity | 25% — the fraction of F2 hybrids expected to be BLIND, which is why F2 should not be used — “We focused on first generation (F1) hybrids to minimize genetic differences between individuals. While further breeding is expected to increase intersubject genetic variability, F2 hybrids will necessarily express recessive traits not expressed by the F1 generation. For instance, 25% of the F2 hybrids are expected to be blind because of a functional copy of the Pde6b gene ( Pittler and Baehr, 1991 ) maintained in every F1 hybrid. Thus, unless careful backcrossing is performed, F2 hybrids should not be used.” | content_tags.quantities[10] |
| quantity | 0 [0 0] — head turns made by FVB in the optomotor drum - the measurement that says one progenitor cannot see — “In contrast, FVB did not make any head turns (0 [0 0]; C57 vs FVB: p = 0.0011; HYB vs FVB: p = 0.003), consistent with blindness because of retinal degeneration ( Pittler and Baehr, 1991 ).” | content_tags.quantities[11] |
| variable | age group (independent) — “Mice of each strain were divided into two age groups, three and nine months old ( Table 1 ).” | content_tags.variables[1] |
| variable | latency to fall (dependent) — “The duration until the animal fell from the rod (“latency to fall”) was measured.” | content_tags.variables[2] |
| variable | weight — “We found that sedentary HYB were larger than either parental strain. At the age of three months, the median [IQR] weight of HYB was 32 [29.6 34.9] g, compared with 26.9 [25.1 28.1] g for C57 ( p = 9.6 × 10 −10 ; Kruskal–Wallis test, corrected for multiple comparisons; Fig. 1 ; Extended Data Fig. 1-1 A ). Higher weights are beneficial for electrophysiological experiments in freely-moving animals, in which animal size limits the weight of the implanted apparatus.” | content_tags.variables[10] |
| variable | anxiety — “However, HYB spent more time in the open arms than C57, indicating reduced anxiety-like behavior ( p = 0.034; Kruskal–Wallis test; Fig. 2 A ; Extended Data Fig. 1-2 ).” | content_tags.variables[11] |
| variable | balance — “C57 and FVB exhibited increased latency to fall over days, indicative of balance learning (C57: p = 0.03, FVB: p = 0.03, within-strain Kruskal–Wallis test; Extended Data Fig. 1-3 C ). HYB latency to fall did not increase over days ( p = 0.12, Kruskal–Wallis test). To summarize, at the age of three months, visual behavior, and motor endurance of HYB are similar to those of C57.” | content_tags.variables[12] |
| variable | latency to platform (dependent) — “During the learning days, the time before finding the platform (“latency to platform”) and the total distance traveled were measured.” | content_tags.variables[13] |
| variable | distance traveled (dependent) — “During the learning days, the time before finding the platform (“latency to platform”) and the total distance traveled were measured.” | content_tags.variables[14] |
| variable | number of head turns (dependent) — “The number of head turns (15° movements at the drum speed) and the cumulative duration of head turns were measured.” | content_tags.variables[15] |
| variable | duration of head turns (dependent) — “The number of head turns (15° movements at the drum speed) and the cumulative duration of head turns were measured.” | content_tags.variables[16] |
| variable | trial duration (dependent) — “Trial duration and distance to failure were measured.” | content_tags.variables[17] |
| variable | distance to failure (dependent) — “Trial duration and distance to failure were measured.” | content_tags.variables[18] |
| definition | contra-anxiety index — “The time spent exploring the open arms (“open”) and the time spent exploring the closed arms (“closed”) were used to derive an index (open – closed)/(open + closed) that served as a contraindication to anxiety-like behavior (“contra-anxiety index”).” | content_tags.definitions[0] |
| definition | aging index — “An “aging index” was defined for every parameter as the difference between the median value of that parameter for nine-month-old mice and the median for three-month-old mice of the same strain, divided by the sum.” | content_tags.definitions[1] |
| definition | C57 index — “A “C57 index” was defined in an equivalent manner, where the index was defined as the difference between the median value of the parameter of interest for the tested strain (FVB or HYB) and the median value of the same parameter for C57 mice of the same age group, divided by the sum.” | content_tags.definitions[2] |
| definition | light-induced firing rate gain — “Light-induced firing rate gain was defined as the mean firing rate during illumination, divided by the mean firing rate during baseline (in the lack of illumination on any shank).” | content_tags.definitions[3] |
| definition | precession effect size — “Precession effect size was quantified as the ratio between the fit of spikes to the circular-linear model, R , divided by the median of 300 model fits to randomly permuted phase/position pairs.” | content_tags.definitions[4] |
| definition | successful run — “A successful run was defined when the animal traversed the track without pausing.” | content_tags.definitions[5] |
| definition | FVB — “FVB is an inbred albino strain which carries a recessive allele causing retinal degeneration ( Pittler and Baehr, 1991 ; Taketo et al., 1991 ).” | content_tags.definitions[6] |
| definition | hybrid — “Hybrids inherit one allele from the C57BL/6J parent, maintaining transgenic properties in a heterozygous manner.” | content_tags.definitions[7] |
| definition | HYB — “We bred FVB/NJ females and C57BL/6J males to generate first-generation hybrid offspring (FVB/NJ x C57BL/6J)F1. The hybrid mice exhibit reduced anxiety-like behavior, improved learning, and enhanced long-term spatial memory. In contrast to both progenitors, hybrids maintain sensorimotor performance upon aging and exhibit improved long-term memory.” | content_tags.definitions[8] |
| definition | strain — “One of the challenges in mouse-based research is adjusting the strain to the specific study question. Strain selection can potentially affect research outcome, since strain characteristics may interact with genetic manipulations ( Sittig et al., 2016 ). Furthermore, strain characteristics could interact with experimental manipulations. For example, mice that suffer from retinal degeneration cannot fully use visual information and exhibit impaired behavior in hippocampal-based navigation tasks that rely on visual cues” | content_tags.definitions[9] |
| key-terms | HYB, C57, FVB, strain, behavior, anxiety, memory, aging, balance, weight, genetic, knock-out, quadrant, reviewer, editor, phenotype, allele, headgear |
| concept | HYB — not defined in this paper's own words; the hybrid, and an abbreviation that is a prefix of the word it abbreviates — “Authors have comparatively tested Hybrid (HYB) mice in the several behavioral paradigms, along with their electrophysiological properties and optogenetic assessments of their neurons.” | concepts[0] |
| concept | C57 — not defined in this paper's own words; the standard strain, and the thing being argued against — “Previous work has shown that offspring of C57BL/6J (C57) and FVB/NJ (FVB) mice consume more alcohol than either progenitor, serving as a preferable model for alcohol consumption ( Blednov et al., 2005 , 2010 ).” | concepts[1] |
| concept | FVB — not defined in this paper's own words; the other progenitor - blind, and chosen anyway — “Previous work has shown that offspring of C57BL/6J (C57) and FVB/NJ (FVB) mice consume more alcohol than either progenitor, serving as a preferable model for alcohol consumption ( Blednov et al., 2005 , 2010 ).” | concepts[2] |
| concept | strain — not defined in this paper's own words; the design decision that decides the outcome — “Phenotyping study design Mice of each strain were divided into two age groups, three and nine months old ( Table 1 ).” | concepts[3] |
| concept | behavior — not defined in this paper's own words; what the strain limits — “One way to balance the requirements of convenient genetic control and complex behavior is to generate hybrids of C57BL/6J and another strain.” | concepts[4] |
| concept | anxiety — not defined in this paper's own words; reduced, which helps some studies and disqualifies others — “For instance, the overall reduced anxiety-like behavior and the age-related changes make the HYB less suitable for long-term studies of anxiety.” | concepts[5] |
| concept | memory — not defined in this paper's own words; improved or unchanged depending on how it is scored — “Open in a new tab Older hybrids exhibit improved learning and long-term memory.” | concepts[6] |
| concept | aging — not defined in this paper's own words; where the whole advantage actually lives, per the reviewer — “I think the manuscript should be clearer regarding that the effect of aging is explaining the better properties of hybrids at relatively old age (9-months) and that hybrids present no major advantage when young adult mice are used (except for their larger size).” | concepts[7] |
| concept | balance — not defined in this paper's own words; the one assay the hybrid loses — “First, compared with the C57 progenitors, HYB exhibited similar or improved performance in every assay used, with one exception: balance performance in the three-month-old age group.” | concepts[8] |
| concept | weight — not defined in this paper's own words; the number that decides what apparatus can be carried — “Higher weights are beneficial for electrophysiological experiments in freely-moving animals, in which animal size limits the weight of the implanted apparatus.” | concepts[9] |
| concept | genetic — not defined in this paper's own words; the control that inbreeding buys — “The hybrid mice allow genetic control while supporting complex behavior over prolonged durations.” | concepts[10] |
| concept | knock-out — not defined in this paper's own words; and the manipulation a robust animal may hide — “Third, because HYB mice are more robust, genetic knock-out manipulations may be less likely to show a consistent phenotypic change ( Sittig et al., 2016 ).” | concepts[11] |
| concept | phenotype — not defined in this paper's own words; the thing that must stay stable under the null — “A second challenge is to ensure the stability of the tested phenotype under the null condition throughout the study period.” | concepts[15] |
| Mann-Whitney U test U | as written: Mann–Whitney’s U test — “Differences between medians of two groups were tested with Mann–Whitney’s U test.” | content_tags.statistics[0] |
| Kruskal-Wallis one-way ANOVA on ranks | as written: Kruskal–Wallis nonparametric analysis of variance — “Differences between medians of three groups or more were tested with Kruskal–Wallis nonparametric analysis of variance and corrected for multiple comparisons using Tukey’s procedure.” | content_tags.statistics[1] |
| two-way ANOVA (nonparametric) | as written: two-way Kruskal–Wallis analysis of variance — “Differences between medians measured along two dimensions (e.g., strain and day) were tested with a two-way Kruskal–Wallis analysis of variance.” | content_tags.statistics[2] |
| Wilcoxon signed-rank test | as written: Wilcoxon’s signed-rank test — “Wilcoxon’s signed-rank test was employed to determine whether a group median is distinct from zero.” | content_tags.statistics[3] |
| exact binomial test | — “To estimate whether a given fraction was smaller or larger than expected by chance, an exact binomial test was used.” | content_tags.statistics[4] |
| likelihood-ratio (G) test of independence G | — “Differences between the proportions of observations of two categorical variables were tested with a likelihood ratio ( G -) test of independence.” | content_tags.statistics[5] |
| Bonferroni correction for multiple comparisons | as written: Bonferroni’s correction — “Bonferroni’s correction was employed in cases of G -test multiple comparisons.” | content_tags.statistics[6] |
| bootstrap | as written: bootstrap procedure — “We used a bootstrap procedure to estimate index dispersion and determine statistical significance.” | content_tags.statistics[7] |
| Kolmogorov-Smirnov normality test | — “Since most tested parameters did not follow a standard normal distribution (Kolmogorov–Smirnov normality test), nonparametric statistical tests were used for all analyses.” | content_tags.statistics[8] |
| significance level α | as written: significance threshold of α = 0.05 — “In all statistical tests used in this study, a significance threshold of α = 0.05 was used.” | content_tags.statistics[9] |
| all descriptive statistics (n, median, IQR, mean, SEM) | as written: n, median, IQR, mean, SEM — “All descriptive statistics (n, median, IQR, mean, SEM) can be found in the results, the figure legends, and the tables.” | content_tags.statistics[10] |
| permutation test | — “The circular-linear model yielded the precession slope, a , and the resultant length of the residuals, R , indicating model fit; statistical significance was determined by a permutation test ( Sloin et al., 2022 ).” | content_tags.statistics[11] |
| Gaussian mixture model (classification) | as written: Gaussian mixture model — “Units were classified into putative pyramidal cells (PYR) or PV-like interneurons (INT) using a Gaussian mixture model ( Stark et al., 2013 ).” | content_tags.statistics[12] |
| Kruskal–Wallis test | — “However, HYB spent more time in the open arms than C57, indicating reduced anxiety-like behavior ( p = 0.034; Kruskal–Wallis test; Fig. 2 A ; Extended Data Fig. 1-2 ).” | content_tags.statistics[13] |
| claim type | method — a second reading reaching the same value; recorded: method — “We bred FVB/NJ females and C57BL/6J males to generate first-generation hybrid offspring (FVB/NJ x C57BL/6J)F1. The hybrid mice exhibit reduced anxiety-like behavior, improved learning, and enhanced long-term spatial memory. In contrast to both progenitors, hybrids maintain sensorimotor performance upon aging and exhibit improved long-term memory.” | alternatives.claim_type |
| method | a battery of standard phenotyping assays run on three strains at two ages, followed by freely-moving recordings and optogenetic manipulation in transgenic hybrids — a second reading DIFFERING from the recorded value; recorded: behavioural-experiment — “Because of genetic tools, mice are increasingly used for neuroscience experiments that were traditionally performed using rats. However, mice are smaller and “less intelligent” than rats, limiting the size of neural implants and the complexity of performed behaviors. Here, we show that these problems are exacerbated by the widespread use of inbred C57BL/6J mice, which are small animals with behavioral and sensorimotor deficits that worsen with age. In line with the established principle of hybrid vigor, we found that first-generation offspring of C57BL/6J and FVB/NJ mice exhibit improved learning and memory, maintain sensorimotor performance on aging, are larger, and exhibit improved behavior while carrying a chronic implant. The hybrid mice allow genetic control while supporting complex behavior over prolonged durations.” | alternatives.method |
| phenomenon | hybrid vigour - improved behaviour and maintained sensorimotor performance on aging in the first-generation cross of two inbred strains — a second reading DIFFERING from the recorded value; recorded: hybrid vigour in laboratory mouse phenotype — “Compared with the parental C57BL/6J progenitor strain, young first-generation hybrid offspring (FVB/NJ x C57BL/6J)F1 exhibited reduced anxiety-like behavior. Upon aging, HYB anxiety-like behavior was further reduced, and learning and long-term memory performance improved. In contrast, learning and memory performance of the parental C57BL/6J and the maternal FVB/NJ progenitor strains did not improve at older age. Furthermore, motor performance of the progenitor strains deteriorated upon aging, whereas HYB sensorimotor abilities were maintained. HYB were larger, ran faster, and performed more trials during electrophysiological experiments on the linear track. HYB and C57 exhibited similar CA1 place cell rate and phase coding. Finally, optogenetic manipulations were readily achieved in transgenic HYB.” | alternatives.phenomenon |
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Hybridterm·def Offspring of C57BL/6J Mice Exhibit Improved Properties for Neurobehavioral Research Hadas E SloinpersName Hadas E SloinpersName 1 Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 6997801, Israel 2 Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel 1, 2, * , Lior BikovskipersName Lior BikovskipersName 3 Myers Neuro-Behavioral Core Facility, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel 4 School of Behavioral Sciences, Netanya Academic College, Netanya 4223587, Israel 3, 4, * , Amir LevipersName Amir LevipersName 1 Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 6997801, Israel 2 Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel 1, 2 , Ortal Amber-VitospersName Ortal Amber-VitospersName 2 Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel 2 , Tomer KatzpersName Tomer KatzpersName 2 Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel 2 , Lidor SpivakpersName Lidor SpivakpersName 1 Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 6997801, Israel 2 Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel 1, 2 , Shirly SomeckpersName Shirly SomeckpersName 1 Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 6997801, Israel 2 Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel 1, 2 , Roni GattegnopersName Roni GattegnopersName 1 Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 6997801, Israel 2 Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel 1, 2 , Shir Sivroni Shir Sivroni 2 Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel 5 Department of Mathematics, Afeka Tel Aviv Academic College of Engineering, Tel Aviv 6910717, Israel 6 Department of Mathematics, The Open University, Raanana 4353701, Israel 2, 5, 6 , Lucas Sjulson Lucas Sjulson 7 Department of Psychiatry and Behavioral Sciences, Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461 7 , Eran Stark Eran Stark 1 Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 6997801, Israel 2 Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel 1, 2, ✉ 1 Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 6997801, Israel 2 Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel 3 Myers Neuro-Behavioral Core Facility, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel 4 School of Behavioral Sciences, Netanya Academic College, Netanya 4223587, Israel 5 Department of Mathematics, Afeka Tel Aviv Academic College of Engineering, Tel Aviv 6910717, Israel 6 Department of Mathematics, The Open University, Raanana 4353701, Israel 7 Department of Psychiatry and Behavioral Sciences, Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461 The authors declare no competing financial interests.
Author contributions: L.B. and E.S. designed research; H.E.S., L.B., A.L., O.A.-V., T.K., L.Sp., S.So., R.G., S.Si., and E.S. performed research; L.B. and E.S. contributed unpublished reagents/analytic tools; H.E.S., L.B., A.L., and E.S. analyzed data; H.E.S., L.B., L.Sj., and E.S. wrote the paper.
This work was supported by the United States-Israel Binational Science Foundation (BSF) Grant 2015577; the European Research Council Grant 679253; the Israel Science Foundation Grant 638/16; the Israel Science Foundation FIRST Program Grant 1871/17; the Rosetrees Trust Grant A1576; the Canadian Institutes of Health Research (CIHR), the International Development Research Centre (IDRC), the Israel Science Foundation (ISF), and the Azrieli Foundation Grant 2558/18; and the Zimin Institute.
* H.E.S. and L.B. contributed equally to this work.
✉ Correspondence should be addressed to Eran Stark at eranstark@tauex.tau.ac.il .
✉ Corresponding author.
Received 2022 Jun 3; Revised 2022 Jun 28; Accepted 2022 Jul 11; Prepublished 2022 Jul 29; Collection date 2022 Jul-Aug.
Copyright © 2022 SloinpersName et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license , which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.
PMCID: PMC9395247 PMID: 35906064 Abstract C57BL/6 is the most commonly used mouse strainterm in neurobehavioral research, serving as a background for multiple transgenic lines. However, C57BL/6 exhibit behavioral and sensorimotor disadvantages that worsen with age. We bred FVBterm/NJ females and C57BL/6J males to generate first-generation hybridterm·def offspring (FVBterm/NJ x C57BL/6J)F1. The hybridterm·def mice exhibit reduced anxiety-like behaviorterm, improved learning, and enhanced long-term spatial memoryterm. In contrast to both progenitors, hybridsterm·def maintain sensorimotor performance upon agingterm and exhibit improved long-term memoryterm. The hybridsterm·def are larger than C57BL/6J, exhibiting enhanced running behaviorterm on a linear track during freely-moving electrophysiological recordings. Hybridsterm·def exhibit typical rate and phase coding of space by CA1 pyramidal cells. Hybridsterm·def generated by crossing FVBterm/NJ females with transgenic males of a C57BL/6 background support optogenetic neuronal control in neocortex and hippocampus. The hybridterm·def mice provide an improved model for neurobehavioral studies combining complex behaviorterm, electrophysiology, and geneticterm tools readily available in C57BL/6 mice.
Keywords: animal models, behaviorterm, freely-moving, FVBterm/NJ, phenotyping, systems neuroscience Significance Statement Because of geneticterm tools, mice are increasingly used for neuroscience experiments that were traditionally performed using rats. However, mice are smaller and “less intelligent” than rats, limiting the size of neural implants and the complexity of performed behaviorsterm. Here, we show that these problems are exacerbated by the widespread use of inbred C57BL/6J mice, which are small animals with behavioral and sensorimotor deficits that worsen with age. In line with the established principle of hybridterm·def vigor, we found that first-generation offspring of C57BL/6J and FVBterm/NJ mice exhibit improved learning and memoryterm, maintain sensorimotor performance on agingterm, are larger, and exhibit improved behaviorterm while carrying a chronic implant. The hybridterm·def mice allow geneticterm control while supporting complex behaviorterm over prolonged durations.
Introduction Many advances in behavioral and biomedical research rely on lab animals. In choosing an animal model, there are always two conflicting considerations. On the one hand, an organism as similar as possible to humans is desired. On the other hand, the least advanced organism for answering the research question is preferred because of ethical considerations. The lab mouse ( Mus musculus ) has emerged as a key model in balancing these demands, sharing multiple physiological systems and genes with humans while exhibiting simple mating. Over the years, numerous inbred mouse strainsterm have been developed, allowing geneticterm modifications, labeling, and manipulation of specific proteins, cells, and organs ( Beck et al., 2000 ). However, there are phenotypic differences between inbred strainsterm used as background for transgenic mice ( Upchurch and Wehner, 1988 ; Võikar et al., 2001 ; Wahlsten et al., 2003 ; Brown and Wong, 2007 ; O’Leary et al., 2011 , 2013 ; Kafkafi et al., 2017 ). Hence, mouse strainterm selection is essential when designing a scientific project.
The mouse strainterm used most often in neurobehavioral research is C57BL/6. The C57BL/6 Jackson Laboratory substrain, C57BL/6J, provided the first extensively sequenced mouse genome ( Mouse Genome Sequencing Consortium, 2002 ) and is among the most widely used inbred strainsterm ( Altman and KatzpersName, 1979 ; Mekada et al., 2009 ). An advantage of inbreeding is that differences between individuals of the same strainterm are minimal. The geneticterm similarity of inbred animals is especially useful for knock-outterm·mth studies, which may require homozygous animals ( Silva et al., 1997 ). Indeed, a large variety of transgenic mice on the C57BL/6 background is available. However, inbreeding exposes undesired phenotypical traits because of homozygous recessive allelesterm and does not guarantee stability over generations because of geneticterm drift ( Brekke et al., 2018 ). C57BL/6-derived mice exhibit known phenotypic disadvantages, including sensitivity to pain, addiction, impaired balanceterm, age-dependent hearing loss, and increased anxietyterm ( Crawley, 1996 ; Mogil et al., 1999 ; Ouagazzal et al., 2006 ). Thus, the behavioral tasks studied using C57BL/6 are limited.
One way to balanceterm the requirements of convenient geneticterm control and complex behaviorterm is to generate hybridsterm·def of C57BL/6J and another strainterm. Hybridsterm·def inherit one alleleterm from the C57BL/6J parent, maintaining transgenic properties in a heterozygous manner. Previous work has shown that offspring of C57BL/6J (C57term) and FVBterm/NJ (FVBterm) mice consume more alcohol than either progenitor, serving as a preferable model for alcohol consumption ( Blednov et al., 2005 , 2010 ). FVBterm is an inbred albino strainterm which carries a recessive alleleterm causing retinal degeneration ( Pittler and Baehr, 1991 ; Taketo et al., 1991 ). The C57term and FVBterm strainsterm have distinct genealogies ( Beck et al., 2000 ; Mekada et al., 2009 ), increasing geneticterm heterogeneity of the hybridterm·def offspring. Thus, hybridsterm·def may serve as a potentially favorable animal model.
Here, we bred C57term males with FVBterm females to determine whether the first-generation hybridsterm·def (HYBterm) are a preferable model for neurobehavioral studies. We tested mice of the three strainsterm (C57term, FVBterm, and HYBterm) using a battery of standard phenotyping assays ( Crawley, 2008 ; Fuchs et al., 2011 ). Compared with either inbred strainterm, HYBterm exhibited similar sensorimotor performance, reduced anxiety-like behaviorterm, faster learning, and improved memoryterm, which were maintained or further improved on agingterm. HYBterm mice were physically larger, and during electrophysiological recordings exhibited enhanced running on a linear track compared with C57term, while CA1 neurons had similar place coding properties. Furthermore, transgenic HYBterm supported optogenetic neuronal control in neocortex and CA1. Together, the results suggest that the hybridterm·def mice constitute a preferable model for systems neuroscience studies combining behavioral tests, electrophysiology, and geneticterm targeting.
Materials and Methods Experimental model and subject details A total of 176 freely-moving adult mice were used in this study. A total of 170 male mice were used for phenotyping, of which 61 were C57BL/6J (C57term; JAX #000664, The Jackson Laboratory); 52 were FVBterm/NJ (FVBterm; JAX #001800); and 57 were hybridterm·def (HYBterm; Table 1 ), offspring of an FVBterm female and a C57term male. Six transgenic mice were used for electrophysiological recordings (Extended Data Fig. 5-1 ). One was single-transgenic and hybridterm·def, generated by crossing an FVBterm female with a parvalbumin (PV)-Cre male (#008069). Two were dual-transgenic, generated by crossing CaMKII-Cre females (JAX #005359) with Ai32 males (#012569). One was dual-transgenic, generated by crossing a PV-Cre female with an Ai32 male. Two were dual-transgenic and hybridterm·def, generated by crossing FVBterm females with second-generation (CaMKII-Cre x Ai32) males. All mice were bred in-house. After separation from the parents, animals were housed in groups of same-litter siblings. Animals were held on a reverse dark/light cycle (dark phase, from 8 A.M. until 8 P.M.). Data recorded from CA1 during linear track behaviorterm were used in a previous report ( SloinpersName et al., 2022 ). All animal handling procedures were in accordance with Directive 2010/63/EU of the European Parliament, complied with Israeli Animal Welfare Law (1994), and approved by the Tel Aviv University Institutional Animal Care and Use Committee (IACUC #01-16-051, #01-19-017, and #01-21-051).
Table 1.
Mice used for phenotyping assays C57term, 3 M FVBterm, 3 M HYBterm, 3 M C57term, 9 M FVBterm, 9 M HYBterm, 9 M Total Battery A 12 12 11 19 14 19 87 Elevated plus-maze 8 8 7 19 14 19 Open field 7 8 7 19 14 15 Rotarod a 4 4 4 19 14 19 Treadmill 8 8 7 19 14 19 Optomotor drum 8 8 7 19 14 19 Forced swim test 6 8 7 19 14 19 Phenotyping cages b - - - 8 8 6 Battery B 10 9 12 20 17 15 83 Weightterm c - - - 20 17 15 Gait analysis 10 9 11 19 12 15 MWM 10 9 11 18 11 15 Phenotyping cages b - - - 8 7 5 Total 22 21 23 39 31 34 170 Open in a new tab a Due to errors in the initial testing of three-month-old mice on the rotarod, an additional replacement cohort of 12 animals was used.
b Mice tested in the phenotyping cages were a subset of the animals used in test Batteries A and B.
c Mice were weighed at least once every week from the age of three months until subjected to test Battery B at nine months of age.
Phenotyping study design Mice of each strainterm were divided into two age groupsterm·var, three and nine months old ( Table 1 ). Each age groupterm·var was further divided into two subgroups: one subgroup was tested on test Battery A, and a second subgroup was used for test Battery B. Animals assigned to Battery A were subjected to assays in the following order: elevated plus maze (1 d); open field (1 d); rotarod (5 d); treadmill (3 d); optomotor drum (1 d); and forced swim test (1 d). Animals assigned to Battery B were tested on the catwalk (1 d) and on the Morris water maze (MWM; 5 d). Mice were used in the phenotyping cages at least a week postbattery and were counterbalanced from Batteries A and B (nine-month-old mice only). The division of assays into batteries was done to limit the number of assays that each mouse would perform. The order of assays in each battery was designed so that each battery began with voluntary, low stress eliciting assays. Between assays, mice were given a minimum of 24 h without human interaction. All tests were initiated at beginning of the dark phase (8 A.M.) and were administered to a single mouse at a time. All equipment was thoroughly cleaned with Virusolve before and between trials. Except for the rotarod and treadmill tests, behaviorterm during all tests was recorded using GigE cameras (ac1300-60gm mono, Basler; frame rate, 25 Hz). Commercial software (Ethovision 15XT; Noldus Information Technology; Noldus et al., 2001 ) was used to analyze all video files.
Elevated plus maze The elevated plus maze is used to evaluate anxiety-like behaviorterm based on the natural uneasiness of rodents toward open, elevated fields ( Rodgers and Dalvi, 1997 ; Crawley, 2000 ; Komada et al., 2008 ). The apparatus consists of a four-armed platform resembling a “+” shape, positioned 40 cm above the floor. Two arms are confined by walls (“closed”; L × W × H: 35 × 5 × 15 cm), whereas two other arms are not walled (“open”; 35 × 5 cm). Similar arms face one another. In the test, the mouse was initially placed at the center of the maze facing one of the closed arms and then allowed to move freely for 7 min. The time spent exploring the open arms (“open”) and the time spent exploring the closed arms (“closed”) were used to derive an index (open – closed)/(open + closed) that served as a contraindication to anxiety-like behaviorterm (“contra-anxiety indexterm·def”).
Forced swim test The forced swim test is used to evaluate depressive-like behaviorterm based on induced “behavioral despair” ( Porsolt et al., 1977 ; Castagné et al., 2011 ; Can et al., 2012 ). The apparatus consists of a clear Plexiglas cylinder (24 cm in height, 19 cm in diameter) filled with 16 cm of water at 22°C. In the test, the mouse was placed in the cylinder for 7 min and then moved to a heated cage until the fur dried completely. Typically, the mouse gradually stopped swimming before being removed. Freeze (immobility) duration, defined as the time the mouse remained floating motionless in the water, was measured during the last 5 min as a manifestation of behavioral despair.
Gait analysis The gait analysis apparatus (CatWalk XT, Noldus Information Technology) enables the assessment of voluntary gait and locomotion in mice ( Ängeby-Möller et al., 2000 ; Crowley et al., 2018 ). The apparatus consists of a hardware system with a glass walkway (L × W: 130 × 20 cm), a GigE video camera, and a software package for the quantitative assessment of animal footprints. The walkway is illuminated from above by red light and from the side by green light. The green light is internally reflected within the glass, except at touched points. The walkway is connected to a dark goal box and enclosed by an adjustable tunnel at one end. In the test, mice were placed at the walkway entrance and allowed to run freely. A successful runterm·def was defined when the animal traversed the track without pausing. Every animal was tested until three successful runsterm·def were completed. For every parameter, the average of the three runs was used for analysis.
Morris water maze The MWM is used to evaluate learning and spatial long-term memoryterm based on the natural tendency of rodents to attempt to escape a body of water ( Morris, 1981 , 1984 ; Vorhees and Williams, 2006 ; Bromley-Brits et al., 2011 ). The apparatus is a circular pool (150 cm diameter, 10 cm depth) filled with water and maintained at a temperature of 23°C. A transparent platform (5 × 5 cm) was fixed 1 cm below the water surface at a constant location. The pool was situated in a room containing distal visual cues, and proximal cues were placed on the inner walls of the pool. The mouse was introduced to the pool at different starting points and allowed 60 s to find the platform and additional 15 s to stay on the platform. If the mouse failed to find the platform within 60 s, the animal was guided to the platform and allowed to stay there for 15 s. The animal swam three times every day during four consecutive “learning days.” During the learning days, the time before finding the platform (“latency to platformterm·var”) and the total distance traveledterm·var were measured. Changes between days were used to evaluate learning performance.
On the fifth day of the assay (“probe day”), the platform was removed, and the mouse was allowed to swim for 60 s. The time spent in every quadrantterm was recorded. The fraction of time spent in the “target” quadrantterm (i.e., close to the location of the missing platform), the number of visits to the target quadrantterm, and the latency to platformterm·var were used to evaluate spatial long-term memoryterm performance.
Open field The open field test provides a way to systematically assess motivational behaviorterm by quantifying exploration of a novel environment and general locomotor activity ( Christmas and Maxwell, 1970 ; Prut and Belzung, 2003 ). In the test, the mouse was initially placed in one of the corners of an open-top Plexiglas box (L × W: 50 × 50 cm, raised 40 cm above the floor), and behaviorterm was recorded for 15 min. Activity, defined as the fraction of pixels that changed in the entire arena between consecutive frames (frame rate, 25 Hz), was measured throughout the run duration to evaluate exploratory behaviorterm.
Optomotor visual test The optomotor drum is based on an apparatus developed for immobile mice ( Mitchiner et al., 1976 ) and allows assessing visual behaviorterm in freely-moving mice ( Abdeljalil et al., 2005 ). The apparatus consists of an elevated stationary platform (20 cm) surrounded by a drum (internal diameter, 39 cm) with vertically oriented black and white stripes on the inside, each spanning 10°. In the test, the mouse was initially habituated to the platform for 2 min, with the drum stationary. The drum then rotated at 2 rpm counterclockwise for 2 min, stopped for 30 s, and rotated clockwise for 2 min. The number of head turnsterm·var (15° movements at the drum speed) and the cumulative duration of head turnsterm·var were measured.
Phenotyping cages The PhenoTyper (model 3000, Noldus Information Technology) is an instrumented home cage in which rodent behaviorterm is automatically monitored through a video-based and event-based system ( De Visser et al., 2006 ; Maroteaux et al., 2012 ; Grieco et al., 2021 ). The cage (L × W × H: 30 × 30 × 35 cm) is made of transparent Plexiglas walls with an opaque Plexiglas floor. The cage is equipped with a watering station, a feeding station, a running wheel, and a shelter in one corner (H × D: 10 × 9 cm, transparent material). The feeding and watering stations are equipped with beam-breaking devices, allowing automatic recording of feeding behaviorterm and water intake (number of feeds and licks). The lid of the cage lid is equipped with an infrared-sensitive video camera (768 × 576 pixels) and several infrared LEDs, allowing continuous recording of animal position. Bedding covered the floor, and food and water were provided ad libitum . The mouse was introduced to the cage at the beginning of the dark phase (8 A.M.). Testing lasted 3 d, during which no human interference took place.
Rotarod test Mice were subjected to a five-lane accelerating rotarod (Ugo Basile) to evaluate motor learning and balanceterm ( Jones and Roberts, 1968 ; Crawley, 2000 , 2003 ; Shiotsuki et al., 2010 ). The apparatus consists of a 3.2 cm (diameter) horizontal rod elevated 10 cm from the ground. The paradigm was comprised of five consecutive days. On every day, mice were subjected to five trials, of which the duration of the three longest trials were averaged. A trial began with the rod rotating at 4 rpm and gradually accelerating for 5 min, up to a maximum of 50 rpm. The duration until the animal fell from the rod (“latency to fallterm·var”) was measured.
Treadmill The treadmill apparatus (Panlab, Harvard Apparatus) is used to assess maximal endurance in mice ( Marques-Aleixo et al., 2015 ). The apparatus consists of a five-lane motorized treadmill with an electric shock zone at one end of the track. Each lane is 38 × 7 × 7 cm (L × W × H). A current shock (0.2 mA) was used to encourage running; cumulative shock duration of 2 s defined the maximal ability of mice to run. The protocol included two training days and one testing day. On the first training day, treadmill speed was fixed at 5 cm/s. The second training day consisted of two parts: (1) walking on the treadmill for 300 s at a constant speed (5 cm/s); (2) subjecting the mice to 330 s of locomotion at a variable speed (increasing from 5 to 21 cm/s by 1 cm/s every 20 s). On the third (testing) day, speed was initially set to 5 cm/s and was gradually increased by 1 cm/s every 20 s. The trial ended when the cumulative shock duration was 2 s. Trial durationterm·var and distance to failureterm·var were measured.
Indices for behavioral assays An “aging indexterm·def” was defined for every parameter as the difference between the median value of that parameter for nine-month-old mice and the median for three-month-old mice of the same strainterm, divided by the sum. We used a bootstrap procedureterm·stat to estimate index dispersion and determine statistical significance. The null hypothesis of the index being equal to zero is equivalent to no consistent changes between the two age groupsterm·var. In the procedure, we resampled the data (e.g., 12 values from three-month-old mice and 15 values from nine-month-old mice) with replacement many (10,000) times. For each resampling iteration, we computed the aging indexterm·def. The reported index is the mean over all iterations, and the SEM is the SD over all iterations. For a one-sided alternative hypothesis (increase/decrease), p -values are the fraction of indices below/above zero.
A “C57 indexterm·def” was defined in an equivalent manner, where the index was defined as the difference between the median value of the parameter of interest for the tested strainterm (FVBterm or HYBterm) and the median value of the same parameter for C57term mice of the same age groupterm·var, divided by the sum. Mean, SEM, and p -values were estimated using bootstrapping as for the agingterm indices.
When parameters are strictly positive, the difference divided by the sum, (a – b)/(a + b), provides a valid estimate. However, some parameters of interest can take negative values, for instance, the “contra-anxiety indexterm·def” ranges from −1 to 1. Then, an increase from one negative value to another will yield a negative “aging indexterm·def” (or “C57 indexterm·def”). Thus, when the parameter of interest was itself an index, we replaced the ratio (a – b)/(a + b) with the a/b ratio.
Probes and surgery Every animal used in electrophysiological experiments (Extended Data Fig. 5-1 ) was implanted with a multi-shank silicon probe attached to a movable microdrive, equipped with optical fibers following previously described procedures ( Stark et al., 2012 ; Noked et al., 2021 ). The probes used were Stark64 (Diagnostic Biochips), Buzaski32 (NeuroNexus), and Dual-sided64 (Diagnostic Biochips). The Stark64 probe consists of six shanks, spaced horizontally 200 μm apart, with each shank consisting of 10–11 recording sites spaced vertically 15 μm apart. The Buzaski32 probe consists of four shanks, spaced horizontally 200 μm apart, with each shank consisting of eight recording sites spaced vertically 20 μm apart. The Dual-sided64 probe consists of two dual-sided shanks, spaced horizontally 250 μm apart, with each shank consisting of 16 channels on each side (front and back), spaced vertically 20 μm apart.
Before probe implantation, the single-transgenic hybridterm·def was injected with a DIO-hChR2 viral vector (rAAV5/EF1a-DIO-hChR2(H134R)-eYFP; 3.2 × 10 12 IU/ml; University of North Carolina viral core facility; courtesy of K. Deisseroth). The solution was injected stereotactically (Kopf) into the neocortex and hippocampus at 8 different depths (AP −1.6, ML 1.1, DV 0.4–1.8 at 0.2 mm increments; 25 nl/site; Nanoject III, Drummond).
Probes were implanted in the parietal neocortex above the right hippocampus (AP/ML, −1.6/1.1 mm; 45° angle to the midline) under isoflurane (1%) anesthesia. Following recovery from anesthesia, linear-track animals were placed on a water-restriction schedule that guaranteed at least 40 ml/kg of water (corresponding to 1 ml/25 g mouse) on every recording day. Recordings were conducted 5 d/week, and animals received free water on the sixth day. After every one to five recording sessions, the probe was translated vertically downwards by up to 70 μm.
Histology After recordings have ended, the implanted mice were deeply anesthetized with pentobarbital (100 mg/kg) and perfused with 0.1 m phosphate buffered saline (PBS; pH 7.4) and 4% paraformaldehyde (PFA). The brains were removed and postfixed overnight in PFA. Coronal sections (70 μm) were cut on a vibratome (VT1000S, Leica) and collected in PBS. Sections were mounted in Fluoromount with DAPI (F6057-20ML, Sigma) and imaged with a wide-field fluorescence microscope (Axio Scope A1, Zeiss).
Linear track sessions For the linear track analyses, neuronal activity was recorded in 4.5 [1.9 10.1]-h sessions (median [interquartile interval, IQR]). At the beginning of every session, neural activity was recorded while the animal was in the home cage. The animal was then placed on a 150 cm linear track that extended between two 10 × 10 cm square platforms. Each platform included a water delivery port. Mice were under water restriction and were trained to repeatedly traverse the track for a water reward of 3–10 μl. Over all sessions, mice ran 167 [132 200] one-direction trials over about 1 h (Extended Data Fig. 5-2 ). Trials with a mean running speed below 10 cm/s were excluded from analyses. Animals were equipped with a three-axis accelerometer (ADXL-335, Analog Devices) for monitoring head movements. Head position and orientation were tracked in real-time using two head-mounted LEDs, a machine vision camera (ace 1300-1200uc, Basler), and a dedicated system (“Spotter,” Gaspar et al., 2019 ).
Spike detection and sorting Neural activity was filtered, amplified, multiplexed, and digitized on the headstage (0.1–7500 Hz, x192; 16 bits, 20 kHz; RHD2132 or RHD2164, Intan Technologies) and then recorded by an RHD2000 evaluation board (Intan Technologies). Offline, spikes were automatically detected and sorted into single units using KlustaKwik3 ( Kadir et al., 2014 ; Rossant et al., 2016 ) for shanks with up to 11 sites/shank or KiloSort2 ( Pachitariu et al., 2016 ) for 16 channel shanks. Automatic spike sorting was followed by manual adjustment of the clusters. Only well-isolated units were used for further analyses [amplitude >40 μV; L-ratio <0.05 ( Schmitzer-Torbert et al., 2005 ); ISI index <0.2 ( Fee et al., 1996 )]. Units were classified into putative pyramidal cells (PYR) or PV-like interneurons (INT) using a Gaussian mixture modelterm·stat ( Stark et al., 2013 ).
Place field and phase precession analysis Based on the linear track data, spatial information, place fields, and phase precession were determined for every PYR ( SloinpersName et al., 2022 ). Briefly, place fields were defined as regions spanning 15–100 cm in which the firing rate increased compared with the on-track spontaneous firing rate ( p < 0.05, Bonferroni-corrected Poisson test). Theta phase precession was quantified for each place field using a circular-linear analysis ( Schmidt et al., 2009 ; Kempter et al., 2012 ). The circular-linear model yielded the precession slope, a , and the resultant length of the residuals, R , indicating model fit; statistical significance was determined by a permutation testterm·stat ( SloinpersName et al., 2022 ). Precession effect sizeterm·def was quantified as the ratio between the fit of spikes to the circular-linear model, R , divided by the median of 300 model fits to randomly permuted phase/position pairs.
Optogenetic stimulation To determine the effect of optogenetic stimulation on spiking, 50-ms blue-light pulses were administered. In every session, illumination was conducted for every shank separately. In CA1, pulses were given in a total of 131 shanks during 55 sessions, using light power of 2.43 [0.96 3.98] μW. In a given stimulation experiment, pulses were applied 137 [51 209] times. In the neocortex (Extended Data Fig. 6-3 ), pulses were given in a total of 63 shanks during 21 sessions, using light power of 11.07 [5.25 21.71] μW. In a given stimulation experiment, pulses were applied 150 [75 300] times. Light-induced firing rate gainterm·def was defined as the mean firing rate during illumination, divided by the mean firing rate during baseline (in the lack of illumination on any shank). Units were determined as light-activated if the Poisson probability of seeing the observed number of spikes (or more) during illumination was < 0.05, based on the baseline firing.
Statistical analyses In all statistical tests used in this study, a significance threshold of α = 0.05 was used. All descriptive statistics (n, median, IQR, mean, SEM)term·stat can be found in the results, the figure legends, and the tables. Since most tested parameters did not follow a standard normal distribution (Kolmogorov–Smirnov normality testterm·stat), nonparametric statistical tests were used for all analyses. All statistical analyses were conducted in MATLAB (MathWorks). Differences between medians of two groups were tested with Mann–Whitney’s U testterm·stat. Differences between medians of three groups or more were tested with Kruskal–Wallis nonparametric analysis of varianceterm·stat and corrected for multiple comparisons using Tukey’s procedure. Differences between medians measured along two dimensions (e.g., strainterm and day) were tested with a two-way Kruskal–Wallis analysis of varianceterm·stat. Wilcoxon’s signed-rank testterm·stat was employed to determine whether a group median is distinct from zero. To estimate whether a given fraction was smaller or larger than expected by chance, an exact binomial testterm·stat was used. Differences between the proportions of observations of two categorical variables were tested with a likelihood ratio ( G -) test of independenceterm·stat. Bonferroni’s correctionterm·stat was employed in cases of G -test multiple comparisons.
Results Hybridterm·def mice are larger than C57term mice and exhibit similar sensorimotor performance We decided to use offspring of C57term and FVBterm because the two inbred strainsterm are of different genealogies ( Beck et al., 2000 ) and exhibit distinct phenotypic disadvantages ( Crawley, 2008 ). FVBterm dams produce large litters ( Silver, 1995 ) and were therefore selected as the maternal strainterm. To maximize the geneticterm similarity between individual mice, we focused on (FVBterm/NJ x C57BL/6J)F1, the first-generation offspring (HYBterm). We found that sedentary HYBterm were larger than either parental strainterm. At the age of three months, the median [IQR] weightterm of HYBterm was 32 [29.6 34.9] g, compared with 26.9 [25.1 28.1] g for C57term ( p = 9.6 × 10 −10 ; Kruskal–Wallis testterm·stat, corrected for multiple comparisons; Fig. 1 ; Extended Data Fig. 1-1 A ). Higher weightsterm are beneficial for electrophysiological experiments in freely-moving animals, in which animal size limits the weightterm of the implanted apparatus. Examination of spontaneous home cage behaviorterm (Extended Data Fig. 1-1 B ) and gait analysis (Extended Data Fig. 1-1 C ) revealed consistent differences between the three strainsterm. Thus, the HYBterm constitute a unique, larger strainterm.
Figure 1.
Open in a new tab Hybridterm·def mice weigh more than both progenitors. Mice of the three strainsterm were weighted periodically at ages between 75 and 105 d (∼3-month-old; A ) or between 255 and 285 d (∼9-month-old; B ), and weightsterm were averaged over measurements for every mouse. Every box plot shows median and interquartile range (IQR), whiskers extend for 1.5 times the IQR in every direction, and a plus indicates an outlier. P -value indicated by text is for a one-way Kruskal–Wallis testterm·stat; *** p < 0.001, Kruskal–Wallis testterm·stat, corrected for multiple comparisons. See also Extended Data Figures 1-1 , 1-2 , and 1-3 .
Extended Data Figure 1-1 Hybridsterm·def are larger and exhibit distinct patterns of daily conduct in the home cage. A , HYBterm weigh more than both progenitors. Top, Mice of the three strainsterm were weighed periodically over six months, from three to nine months old ( n = 20 C57term, 17 FVBterm, and 15 HYBterm). Each line connects measurements of a single animal. P -values indicated by text are for a strainterm by day two-way Kruskal–Wallis testterm·stat. Dashed lines, age ranges used for statistical analyses (center and bottom panels). Every box plot shows median and interquartile range (IQR), whiskers extend for 1.5 times the IQR in every direction, and a plus indicates an outlier. For every mouse, weightsterm were averaged over measurements made in the age of 75–105 d (“3 months”; center) and 255–285 d (“9 months”; bottom); *** p < 0.001, Kruskal–Wallis testterm·stat, corrected for multiple comparisons. B , Hybridsterm·def are least active and spend more time in the shelter during the dark phase. Nine-month-old mice ( n = 16 C57term, 15 FVBterm, and 11 HYBterm) were placed in individual phenotyping cages and the distance covered, time in the shelter, feeding, and licking were sampled every hour over 3 d. a , Top, During both dark (8 A.M. until 8 P.M.) and light phases, HYBterm cover less distance. Bands show SEM. Second row, During the dark phase, HYBterm spend more time in the shelter than C57term or FVBterm. Third row, During both dark and light phases, HYBterm feed less than either C57term or FVBterm. Bottom, The number of licks is not consistently different between the strainsterm. b , Mean metric (distance covered, sheltering, feeding/licking rate) during the dark (top half) and light (bottom) phases. Error bars, SEM; ** p < 0.01, *** p < 0.001, Kruskal–Wallis testterm·stat, corrected for multiple comparisons. c , Diurnal indices, defined as the difference between the metric (e.g., distance covered) during the light phase (L) and the dark phase (D), divided by the sum; * p < 0.05, *** p < 0.001, bootstrapterm·stat test. Mice of all strainsterm exhibit diurnal changes in all metrics tested. d , Pie charts show the fraction of time spent by mice of every strainterm carrying three specific behaviorsterm: sheltering (being in the shelter), feeding (breaking the photobeam of the feeding station), and wheeling (being on the wheel). “Other” corresponds to home cage roaming, including licking. The time mice dedicated to each behaviorterm differs between strainsterm (*** p < 0.001, strainterm, behaviorterm, and interaction effects, two-way Kruskal–Wallis testterm·stat). Compared to the dark phase, mice of all strainsterm spend less time on the wheel and feeding during the light phase (*** p < 0.001, Kruskal–Wallis testterm·stat). C , Gait patterns of the three strainsterm. a , Gait regularity does not differ consistently between strainsterm or between age groupsterm·var. Here and in all other panels, p -values indicated in text in the top and center are for Kruskal–Wallis testterm·stat (e.g., 0.93). Bottom, Agingterm indices; * p < 0.05, *** p < 0.001 bootstrapterm·stat test. b , The front paws base of support (BOS) of HYBterm increases with agingterm. c , The hind paws BOS is distinct for the three strainsterm; * p < 0.05, ** p < 0.01, *** p < 0.001, Kruskal–Wallis testterm·stat, corrected for multiple comparisons. d–f , Track crossing duration, maximum variation, and cadence do not differ consistently between strainsterm in either age groupterm·var. Download Figure 1-1, EPS file (2.1MB, eps) .
Extended Data Figure 1-2 Results of phenotyping assays for every age groupterm·var and strainterm. a Values indicate median [IQR] for every age groupterm·var, strainterm, and metric. For learning on the MWM, values indicate mean ± SEM. Download Figure 1-2, DOCX file (31.1KB, docx) .
Extended Data Figure 1-3 Hybridterm·def sensorimotor performance is maintained upon agingterm. A , Reaction to visual stimuli on the optomotor drum of HYBterm and C57term is similar and remains intact at an older age. In contrast, FVBterm do not react to rotating visual stimuli at either age groupterm·var. Top row, Three-month-old mice. Center, Nine-month-old mice. Bottom, Agingterm indices ( a ) FVBterm mice do not make any head turns, indicating poor visual abilities. Here and in other panels, P -values indicated in text are for a Kruskal–Wallis testterm·stat; ** p < 0.01, *** p < 0.001, Kruskal–Wallis testterm·stat, corrected for multiple comparisons. b , Directional bias in the optomotor drum. Every trial included 2 min of 2 rpm counterclockwise (CCW) and 2 min of 2 rpm clockwise (CW) drum rotations. Directionality indices (DI) were defined as the number of head turnsterm·var during CCW versus CW drum rotations. c , Time spent making head movements. FVBterm mice do not move their heads as the drum turns, whereas the time spent moving the heads does not differ consistently between C57term and HYBterm in either age groupterm·var. B , Motor endurance deteriorates for older C57term and FVBterm mice. Center, At the age of nine months, HYBterm cover longer distances than FVBterm; ** p < 0.01, Kruskal–Wallis testterm·stat, corrected for multiple comparisons. Bottom, In contrast to C57term and FVBterm, HYBterm maintain performance between the two age groupsterm·var. Bars, mean aging indexterm·def; error bars, SEM; ** p < 0.01, *** p < 0.001, bootstrapterm·stat test compared to a no-change null. C , Balanceterm performance deteriorates upon agingterm for C57term and FVBterm. In contrast, HYBterm balanceterm performance is maintained upon agingterm. Top and center, Mean (SEM) time to fall on five consecutive training days; *** next to the text indicate p < 0.001 for a two-way, strainterm by day, Kruskal–Wallis testterm·stat. */*** at right: p < 0.05/p < 0.001, one-way Kruskal–Wallis testterm·stat, measuring within-strain, across-day effect. In the older age groupterm·var, HYBterm remained on the apparatus for the longest duration before falling. Bottom, Agingterm indices were computed for each strainterm every day; * p < 0.05, *** p < 0.001, bootstrapterm·stat test. Inset, Agingterm indices, averaged over days. Download Figure 1-3, EPS file (869.3KB, eps) .
To assess the sensorimotor capabilities of HYBterm, we subjected three-month-old mice to three standard assays ( n = 12 C57term, 12 FVBterm, and 11 HYBterm; Table 1 ; Extended Data Fig. 1-2 ). Visual behaviorterm was quantified in an optomotor drum test ( Abdeljalil et al., 2005 ), in which mice follow vertical visual stimuli by making directional head movements. C57term and HYBterm made similar numbers of head turns (C57term: 7.5 [3 11]; HYBterm: 7 [5 10], p = 0.98, Kruskal–Wallis testterm·stat; Extended Data Fig. 1-3 A ). In contrast, FVBterm did not make any head turns (0 [0 0]; C57term vs FVBterm: p = 0.0011; HYBterm vs FVBterm: p = 0.003), consistent with blindness because of retinal degeneration ( Pittler and Baehr, 1991 ). Motor endurance was tested by running the mice on a treadmill at gradually increasing speed, and measuring the distance to failureterm·var ( Marques-Aleixo et al., 2015 ). HYBterm stayed on the treadmill for 222 [30 262] s, not consistently different from C57term (227 [148 242] s; p = 0.21; Kruskal–Wallis testterm·stat) or from FVBterm (120 [73 197] s; p = 0.65; Extended Data Fig. 1-3 B ). Balanceterm was assessed by placing the mice on an accelerating rotarod for five consecutive days, and measuring the latency to fallterm·var ( Jones and Roberts, 1968 ). C57term and FVBterm exhibited increased latency to fallterm·var over days, indicative of balanceterm learning (C57term: p = 0.03, FVBterm: p = 0.03, within-strain Kruskal–Wallis testterm·stat; Extended Data Fig. 1-3 C ). HYBterm latency to fallterm·var did not increase over days ( p = 0.12, Kruskal–Wallis testterm·stat). To summarize, at the age of three months, visual behaviorterm, and motor endurance of HYBterm are similar to those of C57term.
Hybridterm·def mice exhibit reduced anxietyterm and improved learning and memoryterm To characterize neuropsychiatric phenotypesterm and cognitive performance, we tested three-month-old mice using five assays ( Table 1 ). Anxiety-like behaviorterm was studied using the elevated plus maze ( Rodgers and Dalvi, 1997 ) and quantified by a “contra-anxiety indexterm·def” that ranges from −1 to 1, defined as time spent in (open – closed)/(open + closed) arms. Indices closer to one indicate that more time was spent in the two “open” arms, corresponding to reduced anxiety-like behaviorterm. C57term spent consistently more time in the closed arms compared with the open arms (−0.59 [−0.66 −0.55]; p = 0.016, Wilcoxon’s signed-rank testterm·stat), as did HYBterm (−0.34 [−0.55 −0.22], p = 0.016; Fig. 2 A ; Extended Data Fig. 2-1 A ). However, HYBterm spent more time in the open arms than C57term, indicating reduced anxiety-like behaviorterm ( p = 0.034; Kruskal–Wallis testterm·stat; Fig. 2 A ; Extended Data Fig. 1-2 ).
Figure 2.
Open in a new tab Young hybridsterm·def exhibit reduced anxietyterm. A , At the age of three months, HYBterm exhibit reduced anxiety-like behaviorterm compared with C57term. Anxiety-like behaviorterm was tested in an elevated plus maze and quantified using a “contra-anxiety indexterm·def,” defined as the time spent in the open minus the time spent in the closed arms, divided by the sum. Lined * p < 0.05, Kruskal–Wallis testterm·stat. Here, and in C , * p < 0.05, ** p < 0.01, Wilcoxon’s signed-rank testterm·stat, comparing to chance level (horizontal dashed line). B , HYBterm exhibit improved learning in the MWM task compared with C57term and FVBterm, as measured by the latency to platformterm·var ( a ) and by the distance traveledterm·var ( b ) over days. Bands show mean and SEM; *** next to the top text indicate p < 0.001 for a two-way, strainterm by day, Kruskal–Wallis testterm·stat; */**/*** next to strainterm names indicate p < 0.05/ p < 0.01/ p < 0.001 for a within-strain, across-day one-way Kruskal–Wallis testterm·stat. C , Spatial long-term memoryterm behaviorterm on the MWM. a , When performance is quantified by the fraction of time spent in the target quadrantterm, there is no consistent difference between the strainsterm. b , When performance is quantified by the fraction of visits to the target quadrantterm, HYBterm exhibit improved performance compared with C57term and to FVBterm. All other conventions are the same as in A . D , Compared with C57term, three-month-old HYBterm exhibit improved anxiety-like behaviorterm, improved exploration, and decreased balanceterm performance, as quantified by the “C57 indexterm·def.” Positive Indices indicate improved performance compared with C57term. Circles (and lines) at vertices show mean (and SEM) indices; filled circles indicate significant changes compared with C57term ( p < 0.05, bootstrapterm·stat test). See also Extended Data Figure 2-1 .
Extended Data Figure 2-1 Hybridsterm·def exhibit reduced anxiety-like behaviorterm, improved learning, and improved memoryterm. A , HYBterm explore the open arms of an elevated plus maze more than C57term mice, particularly at an older age, indicating reduced anxiety-like behaviorterm. Here and in subsequent panels, Top and center, Every box plot shows median and interquartile range (IQR), whiskers extend for 1.5 times the IQR in every direction, and a “+” sign indicates an outlier. Here and in Eb , Ec , P -values indicated by text are for a one-way Kruskal–Wallis testterm·stat; lined * p < 0.05, ** p < 0.01, *** p < 0.001, Kruskal–Wallis testterm·stat, corrected for multiple comparisons; * p < 0.05, ** p < 0.01, *** p < 0.001, Wilcoxon’s signed-rank testterm·stat, comparing indices to chance level. Bottom, Aging indexterm·def. Bars and errors show mean and SEM, respectively; *** p < 0.001, bootstrapterm·stat test, compared to a no-change null (index of zero). B , Depressive-like behaviorterm is not consistently different among HYBterm and C57term mice, and is reduced among older FVBterm mice. C , Open field exploration increases at an older age for all strainsterm. Top, In the three-month-old age groupterm·var, HYBterm were more active in the open field than FVBterm; ** p < 0.01, Kruskal–Wallis testterm·stat, corrected for multiple comparisons. Center, In the nine-month-old group, C57term were more active than FVBterm. Bottom, Mice of all three strainsterm exhibited increased activity upon agingterm, evident as above-zero agingterm indices; ** p < 0.01, *** p < 0.001, bootstrapterm·stat test. D , Learning on the MWM task is improved for HYBterm compared to C57term, and is maintained at older age. a , Mean (and SEM) latency to finding the platform (limited to 60 s). Here and in c , **/*** next to the text indicate p < 0.01/ p < 0.001 in a two-way, strainterm by day, Kruskal–Wallis testterm·stat; */**/*** at right indicate p < 0.05/ p < 0.01/ p < 0.001, one-way Kruskal–Wallis testterm·stat, measuring the within-strain, across-day effect. In both age groupsterm·var. only HYBterm showed consistent across-day learning. Bottom, Agingterm indices, averaged over all learning days. Learning performance of C57term deteriorates at an older age. b , Day-by-day “C57term indices” ( Fig. 1 D ), measuring the change in latency to the platform between FVBterm (blue) or HYBterm (green) and C57term mice. Bands show mean and SEM; * p < 0.05, ** p < 0.01, *** p < 0.001, bootstrapterm·stat test. Insets, Mean (and SEM) C57term indices, averaged over all learning days; p -values, geometric mean over all learning days. c , C57term and HYBterm travel progressively shorter distances over training days, indicating learning. All conventions are the same as in a . E , On the probe day of the MWM task, HYBterm spend the longest time in and the shortest latency to the target quadrantterm. a , Time spent in every quadrantterm; * p < 0.05, ** p < 0.01, *** p < 0.001, Wilcoxon’s signed-rank testterm·stat, comparing time in each quadrantterm to chance level (0.25 of the total time). b , Time spent in the target quadrantterm, for three-month-old (top) and nine-month-old (center) mice. Here and in c , Horizontal dashed lines indicate chance level of 0.25, expected for random exploration; other conventions are the same as in A . Compared to C57term or FVBterm, nine-month-old HYBterm spend a larger fraction of time in the target quadrantterm. Bottom, Agingterm indices. Compared to younger HYBterm, older HYBterm spend more time in the target quadrantterm. c , In both age groupsterm·var, the fraction of visits to the target quadrantterm is highest for HYBterm. d , Latency to the platform location, measured during the probe day, is the shortest for HYBterm. Download Figure 2-1, EPS file (1.4MB, eps) .
Depressive-like behaviorterm was tested using a forced swim test, in which longer freeze duration indicates behavioral despair ( Porsolt et al., 1977 ). Mice of all three strainsterm exhibited similar freeze durations (C57term: 124 [94 135] s; FVBterm: 209 [130 261] s; HYBterm: 201 [85 290] s; p = 0.23, Kruskal–Wallis testterm·stat; Extended Data Fig. 2-1 B ). Motivational behaviorterm was quantified by activity levels in an open field ( Christmas and Maxwell, 1970 ). We did not observe consistent differences between C57term and HYBterm in overall activity in the field (C57term: 0.23 [0.17 0.27] %; HYBterm: 0.28 [0.2 0.31] %; p = 0.36, Kruskal–Wallis testterm·stat; Extended Data Fig. 2-1 C ). Thus, compared with C57term, HYBterm exhibit similar depressive-like behaviorterm and similar motivational behaviorterm.
To assess learning and memoryterm behaviorterm, we trained three-month-old mice on the MWM task ( Morris, 1981 ; n = 10 C57term, 9 FVBterm, and 11 HYBterm; Table 1 ). During four consecutive days, mice were placed in a pool with an invisible submerged platform. Over days, only HYBterm exhibited a decrease in the latency to platformterm·var, indicative of learning (C57term: p = 0.67, FVBterm: p = 0.08, HYBterm: p = 0.03, intrastrain Kruskal–Wallis teststerm·stat; Fig. 2 B ). Pooled over days, the mean ± SEM latency to the platform was shorter for HYBterm (19.3 ± 3.9 s), compared with C57term (21.7 ± 4.3 s) and to FVBterm (41.1 ± 3.8 s; p = 1.7 × 10 −13 , Kruskal–Wallis testterm·stat; Fig. 2 Ba ). Similar results were obtained when examining the distance traveledterm·var ( Terry, 2009 ) instead of the latency to platformterm·var ( Fig. 2 Bb ). Hence, compared with both progenitors, three-month-old HYBterm exhibit improved learning performance.
To assess spatial long-term memoryterm, the mice were tested in the MWM during a “probe” day, without a platform. Mice of the three strainsterm spent similar fractions of time in the target quadrantterm ( p = 0.13, Kruskal–Wallis testterm·stat; Fig. 2 Ca ). However, when spatial long-term memoryterm was quantified by the fraction of visits to the target quadrantterm, HYBterm exhibited the largest fraction of visits to the target quadrantterm, indicative of spatial long-term memoryterm (C57term: 0.28 [0.26 0.27]; FVBterm: 0.21 [0.15 0.3]; HYBterm: 0.36 [0.33 0.44]; p = 0.0014, Kruskal–Wallis testterm·stat; Fig. 2 Cb ). Thus, in contrast to both progenitors, three-month-old HYBterm exhibit spatial long-term memoryterm behaviorterm.
To quantify differences between HYBterm (or FVBterm) and the C57term, we treated the C57term strainterm as a reference and computed “C57term indices” for the eight assays ( Fig. 2 D ). For a given metric, the C57 indexterm·def is defined as the difference between the median value of HYBterm (or FVBterm) and the median value of C57term, divided by the sum. Therefore, a C57 indexterm·def above zero indicates higher values of the metric for the tested strainterm, compared with C57term performance. HYBterm performance was similar to C57term in assays testing visual behaviorterm (mean ± SEM C57 indexterm·def: −0.042 ± 0.19, p = 0.36, bootstrapterm·stat test), motor endurance (−0.06 ± 0.2, p = 0.47), depressive-like behaviorterm (0.21 ± 0.23, p = 0.19), learning (day-averaged latency to platformterm·var; 0.05 ± 0.2, p = 0.25), and spatial long-term memoryterm (time in target quadrantterm; −0.011 ± 0.08, p = 0.47). In contrast, HYBterm exhibited reduced anxiety-like behaviorterm (0.31 ± 0.1, p = 0.011) and increased exploratory behaviorterm (0.089 ± 0.05, p = 0.031). Thus, at the age of three months, HYBterm motor endurance and visual behaviorterm is similar to C57term, whereas the HYBterm exhibit reduced anxiety-like behaviorterm.
Older hybridsterm·def maintain sensorimotor performance and exhibit improved memoryterm To examine the suitability of the HYBterm for long-term studies that require stable behavioral performance, we applied all assays also to nine-month-old mice ( Table 1 ). Akin to the three-month-old HYBterm, nine-month-old HYBterm exhibited reduced anxiety-like behaviorterm compared with C57term (contra-anxiety indexterm·def: C57term: −0.56 [−0.75 −0.46]; HYBterm: −0.15 [−0.27 0.15]; p = 4.1 × 10 −6 ; Kruskal–Wallis testterm·stat; Fig. 3 A ; Extended Data Fig. 2-1 A ). The nine-month-old HYBterm exhibited improved learning compared with C57term, as indicated by shorter latency to platformterm·var (C57term: 33.3 ± 2.9 s; HYBterm: 17.8 ± 3 s; p = 1.7 × 10 −13 , Kruskal–Wallis testterm·stat; Fig. 3 B ; Extended Data Fig. 2-1 D ). Furthermore, nine-month-old HYBterm displayed improved spatial long-term memoryterm compared with C57term, as indicated by a larger fraction of time spent in the target quadrantterm (C57term: 0.3 [0.24 0.4]; HYBterm: 0.55 [0.36 0.62]; p = 0.002, Kruskal–Wallis testterm·stat) and by a larger fraction of visits to the target quadrantterm (C57term: 0.35 [0.27 0.4]; HYBterm: 0.45 [0.36 0.5]; p = 0.013; Kruskal–Wallis testterm·stat; Fig. 3 C ; Extended Data Fig. 2-1 E ). Correspondingly, the C57term indices of HYBterm indicated reduced anxiety-like behaviorterm (mean ± SEM C57 indexterm·def: 0.49 ± 0.1, p = 0.00,001), improved learning (day-averaged latency to platformterm·var; 0.35 ± 0.14, p = 0.002), and improved spatial long-term memoryterm (time in target quadrantterm; 0.3 ± 0.06, p = 0.0003; Fig. 3 D ). The performance of nine-month-old HYBterm was similar to C57term in assays testing visual behaviorterm (−0.031 ± 0.1, p = 0.34, bootstrapterm·stat test), motor endurance (0.14 ± 0.12, p = 0.09), balanceterm (0.04 ± 0.05, p = 0.14), exploration (−0.01 ± 0.023, p = 0.35), and depressive-like behaviorterm (0.04 ± 0.08, p = 0.32). Thus, nine-month-old HYBterm exhibit sensorimotor properties which are similar to nine-month-old C57term, while exhibiting reduced anxiety-like behaviorterm and improved learning and memoryterm performance.
Figure 3.
Open in a new tab Older hybridsterm·def exhibit improved learning and long-term memoryterm. A , At the age of nine months, HYBterm exhibit reduced anxiety-like behaviorterm compared with C57term. All conventions are the same as in Figure 2 A . B , Nine-month-old HYBterm exhibit improved learning in the MWM task compared with C57term and FVBterm, indicated by shorter latency to platformterm·var ( a ) and shorter distance travelled ( b ) over days. All conventions are the same as in Figure 2 B . C , Compared with C57term and FVBterm, nine-month-old HYBterm exhibit improved spatial long-term memoryterm, indicated by larger fraction of time spent in the target quadrantterm ( a ) and by a larger fraction of visits to the target quadrantterm ( b ) during the probe day. All conventions are the same as in Figure 2 C . D , Nine-month-old HYBterm exhibit improved anxiety-like behaviorterm, improved learning, and improved spatial long-term memoryterm behaviorterm compared with C57term. All conventions are the same as in Figure 2 D .
To quantify performance differences between three- and nine-month-old mice of the same strainterm, we defined an “aging indexterm·def” ( Fig. 4 ). For a given performance metric, the aging indexterm·def is the difference between the median value obtained for nine- and three-month-old mice, divided by the sum. Thus, an aging indexterm·def of zero indicates no age-related changes in performance, whereas positive indices indicate age-dependent increases. Visual behaviorterm was maintained across age groupsterm·var for all strainsterm (mean ± SEM agingterm indices: C57term, 0.03 ± 0.17, p = 0.41; FVBterm, not available; HYBterm, 0.04 ± 0.13, p = 0.28, bootstrapterm·stat test; Fig. 4 Aa ). While C57term and FVBterm exhibited reduced motor endurance on agingterm (C57term: −0.41 ± 0.1, p = 0.0001; FVBterm: −0.5 ± 0.13, p = 0.0014), HYBterm endurance was maintained (−0.23 ± 0.22, p = 0.11; Fig. 4 Ab ). Finally, while C57term and FVBterm balanceterm performance declined between age groupsterm·var (C57term: −0.2 ± 0.06, p < 0.05; FVBterm: −0.31 ± 0.09, p < 0.05), HYBterm balanceterm was maintained (0.1 ± 0.13, p = 0.75; Fig. 4 Ac ). Thus, while the motor capabilities of C57term and FVBterm deteriorate upon agingterm, HYBterm motor performance remains stable across the two age groupsterm·var studied.
Figure 4.
Open in a new tab Hybridsterm·def maintain sensorimotor performance and exhibit improved long-term memoryterm on agingterm. A , Sensorimotor behaviorterm remains intact at an older age for HYBterm but not for C57term or FVBterm mice. Positive agingterm indices indicate improvement across three- and nine-month-old mice of the same strainterm. a , Visual behaviorterm of HYBterm and C57term remains intact at an older age, whereas FVBterm do not react to rotating visual stimuli at any age. Here and in B , bars indicate mean; error bars, SEM; * p < 0.05, ** p < 0.01, *** p < 0.001, bootstrapterm·stat test compared with a no-change null. b , Motor endurance is reduced for older C57term and FVBterm and maintained for older HYBterm. c , Balanceterm performance deteriorates for C57term and FVBterm on agingterm and is maintained for older HYBterm. B , Psychiatric-like and cognitive performance across age groupsterm·var. All conventions are the same as in A . a , HYBterm, but not C57term or FVBterm, exhibit reduced anxiety-like behaviorterm upon agingterm. b , Learning performance of C57term, but not FVBterm or HYBterm, deteriorates upon agingterm. c , Spatial long-term memoryterm performance of C57term and FVBterm is maintained upon agingterm, whereas HYBterm memoryterm performance improves. C , Comparison of performance changes in eight behavioral assays using the “aging indexterm·def.” All conventions are the same as in Figure 2 D .
Compared with C57term, three-month-old HYBterm exhibited reduced anxiety-like behaviorterm, improved learning, and improved long-term memoryterm ( Fig. 2 ). We tested whether performance on the three assays was maintained across age groupsterm·var in every strainterm. Compared with three-month-old mice of the same strainterm, anxiety-like behaviorterm was reduced for nine-month-old HYBterm (mean ± SEM aging indexterm·def: 0.26 ± 0.11, p = 0.0005, bootstrapterm·stat test), but not for C57term (0.04 ± 0.12, p = 0.37) or FVBterm (0.22 ± 0.17, p = 0.07; Fig. 4 Ba ). Learning performance was reduced in older C57term mice (−0.28 ± 0.14, p = 0.03), but maintained for older HYBterm (0.04 ± 0.21, p = 0.43; Fig. 4 Bb ). Both C57term and FVBterm maintained spatial long-term memoryterm performance between age groupsterm·var (C57term: 0.008 ± 0.13, p = 0.28; FVBterm: −0.07 ± 0.16, p = 0.16). In contrast, older HYBterm exhibited improved spatial long-term memoryterm (0.31 ± 0.07; p = 0.0003; Fig. 4 Bc ). Thus, anxiety-like behaviorterm is reduced and memoryterm performance improves on agingterm in HYBterm ( Fig. 4 C ).
Hybridterm·def mice exhibit enhanced linear track running and typical CA1 place coding The results reported in Figures 1 - 4 show that HYBterm exhibit certain advantages over C57term. However, improved performance on behavioral assays spanning several minutes does not necessarily predict enhanced behaviorterm over daily recording sessions that span hours and include the additional weightterm of an implanted apparatus. To assess the suitability of freely-moving HYBterm for electrophysiological experiments, we performed high-density recordings from two HYBterm and two C57term mice running back and forth on a 150-cm linear track (Extended Data Fig. 5-1 ). Behaviorterm during the first 14 sessions on the track was assessed to quantify performance at similar experience levels. HYBterm conducted a median [IQR] of 164 [139 215] one-direction trials per session, while C57term ran 136 [113 178] trials ( p = 0.015, Mann–Whitney test; Fig. 5 A ; Extended Data Fig. 5-2 ). Furthermore, HYBterm exhibited higher running speeds, 31 [22 44] cm/s compared with 23 [18 30] cm/s for C57term ( p = 0.007, Mann–Whitney test). Thus, freely-moving HYBterm exhibit enhanced motor abilities and exploration during prolonged electrophysiological recordings.
Figure 5.
Open in a new tab Hybridterm·def mice exhibit enhanced linear track running and typical CA1 place coding. A , Compared with C57term mice, HYBterm carry out more trials and run faster on a 150-cm-long linear track. Only the first 14 sessions from every mouse were employed (two C57term and two HYBterm mice); * p < 0.05, ** p < 0.01, Mann–Whitney test. Dashed vertical lines indicate group medians. a , The number of one-direction trials. b , Mean running speed over trials. B , Location of pyramidal cell (PYR) somata relative to the center of the CA1 pyramidal cell layer (dashed horizontal line) is not consistently different between strainsterm. The estimated depths of PYR recorded by high-density silicon probes were partitioned into 5-μm bins. Positive numbers correspond to PYR closer to str. oriens. n.s.: p > 0.05, Mann–Whitney test. C , Units recorded from both strainsterm exhibit increased firing rates within specific regions of the linear track. Each row represents a unit; firing rates on right (R) to left (L) runs are concatenated with L to R runs and scaled to the 0–1 (white-black) range for presentation purposes. Bar graphs (right) show peak on-track firing rates. D , A PYR recorded from a HYBterm exhibits a typical place field and theta phase precession. Top, Firing rate as a function of position (mean ± SEM over 82 right-to-left trials). Bottom, Theta phase and animal position at the time of every spike; phase of zero corresponds to theta peak. Running direction is presented from left to right, and vertical dashed lines indicate place field limits. a represents phase precession slope, and R represents the goodness of fit of spikes to the precession model; ** p < 0.01, permutation testterm·stat. Left, Wide-band (0.1–7500 Hz) spike waveforms (mean ± SD) recorded on eight consecutive sites, vertically spaced by 20 μm. Bottom left, Autocorrelation histogram. E , HYBterm and C57term exhibit similar CA1 spatial rate coding. a , Spatial information rate. n.s.: p > 0.05, Mann–Whitney test. Here and in c , vertical dashed lines indicate group medians. b , Fraction of CA1 PYR with one or more place fields out of all units active and stable on the track; n.s.: p > 0.05, likelihood ratio test. Here and in Fa , *** p < 0.001, exact binomial testterm·stat, compared with chance level (0.05; horizontal dashed line). Error bars, SEM. c , Place field size; n.s.: p > 0.05, Mann–Whitney test. F , HYBterm and C57term exhibit similar CA1 spatial phase coding. a , Fraction of fields exhibiting theta phase precession; n.s.: p > 0.05, likelihood ratio test. b , Precession slope size. c , Precession effect sizeterm·def; n.s.: p > 0.05, Mann–Whitney test. See also Extended Data Figures 5-1 and 5-2 .
Extended Data Figure 5-1 Mice used for electrophysiological recordings. a For C57term animals, the male parent was the reporter, and the female parent was the driver. b At time of implantation. Download Figure 5-1, DOCX file (31.1KB, docx) .
Extended Data Figure 5-2 Linear track behaviorterm and place coding in individual mice. a Number of one direction trials, median [IQR] over sessions. b Well-isolated PYR, active and stable on the linear track. c SI, spatial information. Download Figure 5-2, DOCX file (31.1KB, docx) .
To examine whether CA1 pyramidal neurons (PYR) exhibit similar place coding in HYBterm and C57term mice tested under the same conditions, we analyzed the activity of CA1 pyramidal layer PYR active on the track (C57term: n = 779, HYBterm: n = 960; Fig. 5 B ; Extended Data Fig. 5-2 ). In both strainsterm, most active PYR exhibited increased firing rates in specific parts of the track ( Fig. 5 C ). An example PYR recorded in HYBterm exhibited typical spatial rate coding (place field; O’Keefe and Dostrovsky, 1971 ; Moser et al., 2008 ) and spatial phase coding (theta phase precession; O’Keefe and Recce, 1993 ; Fig. 5 D ). At the group level, there was no consistent difference in the spatial information carried by PYR, with 1.28 [0.47 3.18] bits/s for HYBterm PYR and 1.37 [0.63 2.87] bits/s for C57term PYR ( p = 0.56, Mann–Whitney test; Fig. 5 Ea ). Place fields were detected in 443/779 (57%) of C57term PYR and in 508/960 (53%) of HYBterm PYR ( Fig. 5 Eb ). Both fractions were above expected by chance ( p < 1.11 × 10 −16 , exact binomial testterm·stat) and were not consistently different from each other ( p = 0.37, likelihood ratio test). Furthermore, place field sizes were not consistently different, spanning 40 [27.5 57.5] cm for C57term PYR and 40 [25 57.5] cm for HYBterm PYR (603 C57term fields and 649 HYBterm fields; p = 1, Mann–Whitney test; Fig. 5 Ec ). Thus, CA1 PYR recorded in HYBterm and in C57term mice exhibit similar rate coding of space.
To determine whether HYBterm CA1 PYR exhibit typical phase coding of space, we examined theta phase precession within PYR place fields. Of all fields, 308/603 (51%) C57term PYR and 353/649 (54%) HYBterm PYR exhibited consistent spatial theta phase precession ( p < 1.11 × 10 −16 , exact binomial testterm·stat; Fig. 5 Fa ). The fraction of place fields with precession was not consistently different between the two strainsterm ( p = 0.51, likelihood ratio test). Precession slope size was not consistently different between strainsterm, being 0.02 [0.01 0.03] cyc/cm for C57term PYR place fields and 0.02 [0.02 0.03] cyc/cm for HYBterm PYR place fields ( p = 0.071, Mann–Whitney test; Fig. 5 Fb ). Finally, precession effect sizeterm·def was not consistently different between strainsterm, with 1.6 [1.28 2.13] for C57term phase precessing fields and 1.54 [1.24 2.06] for HYBterm fields ( p = 0.29, Mann–Whitney test; Fig. 5 Fc ). Thus, CA1 PYR recorded in HYBterm and in C57term exhibit similar phase coding of space.
Transgenic hybridterm·def mice enable optogenetic control of individual neurons A major advantage of C57term mice for neurobehavioral research is the myriad transgenic strainsterm available on the same background. To combine geneticterm control available for C57term with the enhanced behaviorterm of the HYBterm, we first generated dual-transgenic mice on a C57term background by breeding CaMKII-Cre females with Ai32 males. We then crossed the male offspring with FVBterm females. Opsin expression in the resulting dual-transgenic and hybridterm·def mice was verified using histology (Extended Data Fig. 6-1 A ). The dual-transgenic HYBterm are susceptible to blue-light optogenetic activation of projection neurons, including neocortical and CA1 PYR. Optogenetic PYR activation was conducted in transgenic HYBterm implanted with multi-site optoelectronic probes (Extended Data Fig. 5-1 ). A total of 5568 PYR and 1066 putative interneurons (INT) were recorded from the CA1 pyramidal layer during n = 55 sessions (Extended Data Fig. 6-2 ). In every recording session, we tested the response of the recorded units to 50-ms blue-light pulses. During illumination, PYR exhibited increased firing rates ( Fig. 6 A , purple). Of all recorded PYR, 1213 (22%) exhibited a consistent increase in firing rate during illumination ( p < 1.11 × 10 −16 , exact binomial testterm·stat).
Figure 6.
Open in a new tab Transgenic hybridterm·def mice enable optogenetic control of individual CA1 neurons. A , Directly activated PYR and an indirectly activated interneuron (INT) recorded in hippocampal region CA1 of a HYBterm dual-transgenic (CaMKII::ChR2) mouse. a , The units exhibit a cross-correlation histogram (center; no light condition) consistent with monosynaptic excitation ( p < 0.001, Poisson test). b , Local field and spiking responses to a single 50-ms blue-light pulse (0.67 μW). The PYR and the INT both spike during the light. c , Raster plots (top) and PSTHs (bottom) during blue light pulses for the PYR (left) and the INT (right). For visualization purposes, raster plots include 200 pulses. d , Top parts, Mean PSTHs of 22 PYR (purple) and four INT (green) recorded simultaneously on the illuminated shank (S1). Bottom parts, Greyscale, PSTHs of all 48 PYR and eight INT recorded simultaneously during the session. Each row shows the PSTH of one unit, scaled to the 0–1 (white-black) range for visualization purposes. Arrows indicate the example PYR and INT. B , Activation probability of CA1 units depends on the distance from the illuminated shank. Dataset includes a total of 5568 PYR and 1066 INT. a , Bars show the fraction of units with increased firing rate ( p < 0.05, Poisson test) during single-shank illumination. Error bars, SEM; *** p < 0.001, exact binomial testterm·stat, comparing the fraction of light-activated units to chance level (horizontal dashed line). b , Median firing rate gain of activated PYR and INT. Error bars, IQR. Gain is shown only for distances with an above-chance number of optically activated units. P -values indicated in text are for a one-way Kruskal–Wallis testterm·stat; *** p < 0.001, Kruskal–Wallis testterm·stat. See also Extended Data Figures 6-1 , 6-2 , and 6-3 .
Extended Data Figure 6-1 Opsin expression in transgenic and injected hybridsterm·def. A , Opsin expression in neocortex and hippocampus of mA234, a double transgenic and hybridterm·def mouse (FVBterm/NJ x CaMKII-Cre x Ai32) used in linear track ( Fig. 5 ) and optogenetic ( Fig. 6 ) experiments. EYFP fluorescence (green) is used as a reporter for ChR2 expression. Left, Low-magnification (2.5×) image of neocortex and hippocampus of a transgenic mouse. Right, High-magnification (20×) image, showing EYFP in neocortical somata. B , Opsin expression in neocortex and hippocampus of a HYBterm injected with a viral vector (rAVV5/CaMKII-hChR2(H134R)-mCherry) in the right hemisphere. mCherry fluorescence (red) is used as a reporter for ChR2 expression. All other conventions are the same as in A . Download Figure 6-1, EPS file (31.6MB, eps) .
Extended Data Figure 6-2 Optogenetic activation of individual neurons in individual mice. a Well-isolated PYR with increased firing rate during optogenetic stimulation ( p < 0.05, Poisson test), out of all PYR recorded in the same sessions. b Light-activated PYR recorded on the illuminated shank. Download Figure 6-2, DOCX file (31.1KB, docx) .
Extended Data Figure 6-3 Transgenic hybridsterm·def support robust optogenetic activation of individual neocortical neurons. A , B , Optogenetic activation in the neocortex of hybridterm·def freely-moving dual-transgenic CaMKII::ChR2 mice. A , Example directly activated PYR and indirectly activated INT recorded in HYBterm neocortex. a , The units exhibit cross-correlation histogram (center; no light condition) consistent with monosynaptic excitation ( p < 0.001, Poisson test). Bottom, Auto-correlation histograms (ACHs). Wide-band (0.1–7500 Hz) spike waveforms (mean ± SD) recorded on the same shank; intersite vertical spacing is 20 μm. b , Local field and spiking responses of the units to a single 50-ms blue-light pulse (1.71 μW). Both units spike during the light. c , Raster plots (top) and PSTHs (bottom) during 230 blue light pulses for the PYR (left) and indirectly activated INT (right). The raster plot shows 200 representative repetitions. d , Top, Mean PSTHs of 26 PYR (purple) and seven INT (green) recorded simultaneously on the illuminated shank during light pulses. Bottom, Greyscale, PSTH of all unit recorded during the session (49 PYR and 21 INT). S1, S2, S3, and S4 denote different shanks. Each row corresponds to the PSTH of one unit, scaled to the 0–1 (white-black) range for visualization purposes. Arrows indicate the example units. B , Activation probability and firing rate gain of neocortical units depend on the distance from the illuminated shank. Dataset includes a total of 1546 PYR and 517 INT. a , Bars show the fraction of units with increased firing rate ( p < 0.05, Poisson test) during single-shank illumination as a function of the horizontal distance from the illuminated shank. Error bars, SEM; *** p < 0.001, exact binomial testterm·stat, compared to chance level (0.05, horizontal dashed line). Neocortical PYR and INT recorded up to 600 μm away from the illuminated shank are activated. b , Median firing rate gain of activated PYR and INT as a function of distance from the illuminated shank. Error bars, IQR. Gain is considered only for distances with an above-chance number of activated units. P -values indicated in text are for a one-way Kruskal–Wallis testterm·stat; *** p < 0.001, Kruskal–Wallis testterm·stat, corrected for multiple comparisons. Gain differs between activated PYR recorded on the illuminated shank and activated PYR recorded 400 μm away. C , Parvalbumin-immunoreactive (PV) cell activation in a hybridterm·def freely-moving single-transgenic PV-Cre mouse, injected with a Cre-dependent viral vector for ChR2. a , Subnetwork of a PYR (PYR1; purple) and a PV cell (green), recorded simultaneously using a high-density optoelectronic probe. ACHs and cross-correlations (no light condition), consistent with reciprocal monosynaptic excitation and inhibition ( p < 0.001, Poisson test). b , Wide-band (0.1–7500 Hz) traces and spikes of the PV cell and another unit, PYR2. Top three traces are neuronal recordings; fourth trace indicates instantaneous light power emitted by the fiber attached to the recording shank; and the bottom trace indicates head acceleration. During blue light pulses (62 μW), the PV cell spikes, whereas PYR2 spiking is suppressed. c , Raster plot (top) and PSTH (bottom) during blue light pulses for the light-activated PV cell (left) and the suppressed PYR (right). Arrows indicate the example units. d , PSTHs of six units recorded by the illuminated shank during blue light pulses. Bottom, Each row corresponds to the PSTH of one unit, scaled to the 0–1 (white-black) range for visualization purposes. Top, Mean PSTHs. Download Figure 6-3, EPS file (13MB, eps) .
As previously reported in rat hippocampus ( Stark et al., 2012 ), activation probability depended on the horizontal distance from the light source. Of the PYR recorded on the illuminated shank, 772/1527 (47%) exhibited optical activation ( p < 1.11 × 10 −16 , exact binomial testterm·stat), compared with 340/1984 (17%) units recorded on the adjacent shank (horizontal spacing, 200 μm; p = 2.5 × 10 −13 , exact binomial testterm·stat; Fig. 6 Ba , left). The fraction of optically-activated PYR was lower on shanks farther away (0 vs 400 μm away: p < 1.1 × 10 −16 , Bonferroni-corrected likelihood ratio test). Moreover, the firing rate gain of the PYR that responded to focal illumination depended on the distance from the illuminated shank, being 6.5 [3.59 13.97] for same-shank PYR, compared with 4.1 [2.49 7.35] for PYR recorded on the adjacent shank ( p = 9.6 × 10 −10 , Kruskal–Wallis testterm·stat; Fig. 6 Bb , left). Therefore, CA1 PYR in dual-transgenic HYBterm exhibit robust distance-dependent responses to focal illumination.
Optogenetic stimulation led to the indirect activation of INT via monosynaptic inputs from excitatory neurons ( Fig. 6 A , green). A total of 241/1066 (23%) of CA1 INT exhibited light-induced activation ( p < 1.11 × 10 −16 , exact binomial testterm·stat; Fig. 6 Ba , right). The fraction of light activated INT was 34% (92/273) on the illuminated shank and lower on shanks farther away (400 μm: 40/258, 16%, p = 1.3 × 10 −4 , Bonferroni-corrected likelihood ratio test). In contrast to PYR, the firing rate gain of light-activated INT did not differ consistently between shanks ( p = 0.38, Kruskal–Wallis testterm·stat; Fig. 6 Bb , right). Thus, consistent with previous observations in C57term mice ( Stark et al., 2013 ), indirect INT activation in CA1 leads to more widespread activation than the local activation achieved in PYR.
To examine HYBterm optogenetic activation in a brain region with a distinct architecture, we performed optical stimulation in the neocortex of the same mice ( n = 21 sessions; Extended Data Fig. 6-3 ). Similar to CA1 units, neocortical PYR and INT exhibited induced spiking (Extended Data Fig. 6-3 A ), with activation probability that decreased with distance from the illuminated shank (Extended Data Fig. 6-3 Ba ). A total of 274/409 (67%) neocortical PYR recorded on the illuminated shank exhibited optical activation, compared with 722/1527 (47%) CA1 PYR ( p = 1.2 × 10 −4 , likelihood ratio test). Furthermore, 331/591 (56%) neocortical PYR exhibited optical activation on the adjacent shank (200 μm away), compared with 340/1984 (17%) CA1 PYR ( p < 3.5 × 10 −11 , likelihood ratio test). Compared with CA1, focal illumination in the neocortex activated PYR located farther away from the illuminated shank. Light-induced firing rate gainterm·def did not differ between the illuminated shank (7.6 [4.54 16.03]) and the adjacent shank (7.6 [4.08 13.94]; p = 0.79, Kruskal Wallis test; Extended Data Fig. 6-3 Bb , left). The difference between brain regions is consistent with different network topologies, since in contrast to the neocortex, CA1 PYR do not exhibit abundant recurrent excitation ( Thomson and Radpour, 1991 ; Buhl and Whittington, 2007 ). Neocortical units on distant shanks may be activated indirectly via excitatory synaptic connections from directly activated PYR closer to the illumination source.
An alternative approach for establishing optogenetic control in HYBterm is to cross FVBterm females with single-transgenic driver male mice and to inject the offspring with a viral vector that expresses a reporter gene. To demonstrate feasibility, we generated a PV-Cre x FVBterm mouse and injected the animal with a viral vector that allows Cre-dependent expression of ChR2. Focal illumination in the transgenic HYBterm induced direct PV activation and indirect PYR silencing (Extended Data Fig. 6-3 C ). In summary, crossing transgenic C57term with FVBterm yields transgenic HYBterm offspring, suitable for optogenetic experiments.
Discussion Compared with the parental C57BL/6J progenitor strainterm, young first-generation hybridterm·def offspring (FVBterm/NJ x C57BL/6J)F1 exhibited reduced anxiety-like behaviorterm. Upon agingterm, HYBterm anxiety-like behaviorterm was further reduced, and learning and long-term memoryterm performance improved. In contrast, learning and memoryterm performance of the parental C57BL/6J and the maternal FVBterm/NJ progenitor strainsterm did not improve at older age. Furthermore, motor performance of the progenitor strainsterm deteriorated upon agingterm, whereas HYBterm sensorimotor abilities were maintained. HYBterm were larger, ran faster, and performed more trials during electrophysiological experiments on the linear track. HYBterm and C57term exhibited similar CA1 place cell rate and phase coding. Finally, optogenetic manipulations were readily achieved in transgenic HYBterm.
Mice have emerged as a dominant model for biomedical research, mainly because of their small size, high reproductive rate, and geneticterm similarities to humans. The same properties facilitated the engineering of multiple transgenic lines. The maintenance of transgenic lines, particularly the generation of double-transgenic or multitransgenic mice, is greatly facilitated by inbred mice ( Silva et al., 1997 ). However, inbred mice are suboptimal for neurobehavioral studies because of idiosyncratic recessive traits. Furthermore, C57term exhibit age-dependent deterioration, limiting the duration of prolonged experiments. C57term mice are also smaller than other strainsterm, which is a disadvantage for electrophysiological studies involving implanted headgearterm. Since headgearterm weightterm is limited by body size, the range of studied behaviorsterm is limited by animal weightterm. A single well-defined outbreeding step produces larger offspring that exhibit enhanced behaviorterm even during electrophysiology experiments, maintaining the ability of geneticterm targeting.
One of the challenges in mouse-based research is adjusting the strainterm to the specific study question. Strainterm selection can potentially affect research outcome, since strainterm characteristics may interact with geneticterm manipulations ( Sittig et al., 2016 ). Furthermore, strainterm characteristics could interact with experimental manipulations. For example, mice that suffer from retinal degeneration cannot fully use visual information and exhibit impaired behaviorterm in hippocampal-based navigation tasks that rely on visual cues ( Nguyen et al., 2000 ). A second challenge is to ensure the stability of the tested phenotypeterm under the null condition throughout the study period. In many cases, the effects of a given manipulation are studied over weeks to months ( Prevot et al., 2019 ; Namdar et al., 2020 ; Rahn et al., 2021 ). Deterioration of control phenotypic behaviorterm could confound potential manipulation-based intergroup differences. The present work showed that at the age of three months, HYBterm were larger than C57term and exhibited reduced anxiety-like behaviorterm, but sensorimotor performance did not differ. Upon agingterm, C57term sensorimotor capabilities and learning performance deteriorated. In contrast, HYBterm sensorimotor capabilities did not deteriorate on agingterm, and the older HYBterm exhibited reduced anxiety-like behaviorterm and improved long-term memoryterm performance. Thus, HYBterm exhibit particularly stable performance during agingterm, which is potentially useful for long-term studies.
To provide a quantitative description of the HYBterm, we supplemented the array of standard assays with gait assessment and home cage behaviorterm. The gait of all strainsterm was inconsistent with abnormalities seen in motor disorders ( Preisig et al., 2016 ; Rahn et al., 2021 ), yet every strainterm exhibited distinct gait patterns that were largely preserved upon agingterm. In addition, we observed apparent interstrain differences of daily conduct in the home cage, including distinct time division between sheltering, wheel running, and feeding. Together, the weightterm, daily conduct, and gait differences indicate that the spontaneous behaviorterm of the (FVBterm/NJ x C57BL/6J)F1 hybridsterm·def is distinct from that of both progenitor strainsterm.
There are some situations in which hybridterm·def mice are not advantageous. First, compared with the C57term progenitors, HYBterm exhibited similar or improved performance in every assay used, with one exception: balanceterm performance in the three-month-old age groupterm·var. HYBterm impaired balanceterm may result from weightterm differences between the strainsterm since HYBterm weigh more, and weightterm affects rotarod performance ( Mao et al., 2015 ). Although HYBterm balanceterm performance improved upon agingterm, hybridsterm·def may be unsuitable for studies involving balanceterm. Second, while the reduced anxietyterm exhibited by HYBterm is beneficial for studies involving cognitive and motor tasks, reduced anxiety-like behaviorterm may be suboptimal for other purposes. For instance, the overall reduced anxiety-like behaviorterm and the age-related changes make the HYBterm less suitable for long-term studies of anxietyterm. Third, some genes expressed by progenitors may be dominant; for instance, FVBterm contain a Disc1 mutation which has been shown to cause cognitive impairment even when heterozygous ( Koike et al., 2006 ; Ritchie and Clapcote, 2013 ). Nevertheless, we found that the potentially deleterious effects of dominant genes on HYBterm are smaller than the adverse effects of inbreeding. Third, because HYBterm mice are more robust, geneticterm knock-outterm·mth manipulations may be less likely to show a consistent phenotypic change ( Sittig et al., 2016 ). Fourth, it is not straightforward to use hybridsterm·def if a homozygous alleleterm is required or if knock-outterm·mth of both copies of a given gene is required (although see Silva et al., 1997 ).
For studies that do not involve geneticterm manipulations, HYBterm can be used as-is, providing improved performance while maintaining minimal intersubject differences. Specifically, HYBterm can be implanted with high-density electrode arrays for neocortical and hippocampal recordings over multiple months. To combine transgenic control with enhanced behaviorterm, we used two distinct strategies. One simple strategy involves using a homozygous parent of a C57-background, e.g., a driver line, yielding heterozygous HYBterm offspring that present one transgenic alleleterm. For conditional expression of a reporter gene, HYBterm can be injected with a viral vector (as in Extended Data Figs. 6-1 B , 6-3 C ). A second possibility is for one of the parents to be multi-transgenic, e.g., derived by crossing C57-based driver and reporter lines (as in Fig. 6 ; Extended Data Figs. 6-1 A , 6-3 A , B ). Yet a third strategy is to use a C57-based driver line and backcross the reporter onto an FVBterm/NJ background. Because of the large body of transgenic mice developed on the C57term background, the number of possible combinations is very large, allowing to harness the behavioral potential of the hybridsterm·def for a wide range of studies.
We focused on first generation (F1) hybridsterm·def to minimize geneticterm differences between individuals. While further breeding is expected to increase intersubject geneticterm variability, F2 hybridsterm·def will necessarily express recessive traits not expressed by the F1 generation. For instance, 25% of the F2 hybridsterm·def are expected to be blind because of a functional copy of the Pde6b gene ( Pittler and Baehr, 1991 ) maintained in every F1 hybridterm·def. Thus, unless careful backcrossing is performed, F2 hybridsterm·def should not be used. Second, we employed HYBterm which are offspring of FVBterm females and C57term males. Full characterization is required before the use of reciprocal hybridsterm·def, offspring of a C57term female and an FVBterm male. However, because of the reduced litter sizes of female C57term compared with female FVBterm ( Taketo et al., 1991 ; Silver, 1995 ), the usage of the reciprocal hybridsterm·def is not recommended. Finally, since interstrain differences are also sex-dependent ( Brown and Wong, 2007 ), female HYBterm may exhibit distinct phenotypesterm.
To conclude, we showed that a single breeding step produces a hybridterm·def mouse strainterm that exhibits improved behavioral performance while maintaining geneticterm control. The hybridterm·def vigor and the enhanced behavioral capabilities may yield more trials in every experimental session, shorter learning periods, and higher accuracy in complex tasks. By increasing yield and accuracy, the usage of HYBterm may allow uncovering unexplored neuronal mechanisms. Enhanced behaviorterm also has ethical advantages, since faster learning and higher yield per animal translate to fewer experimental animals or sessions required to answer a given question. The combination of geneticterm tools and the enhanced behavioral capabilities of the hybridterm·def mice offers a unique opportunity for studying the neuronal basis of behaviorterm.
Acknowledgments Acknowledgments: We thank Ronni Cohen for constructive comments.
Synthesis Reviewing Editorterm: Mark Laubach, American University Decisions are customarily a result of the Reviewing Editorterm and the peer reviewersterm coming together and discussing their recommendations until a consensus is reached. When revisions are invited, a fact-based synthesis statement explaining their decision and outlining what is needed to prepare a revision will be listed below. The following reviewerterm(s) agreed to reveal their identity: Sebastien Royer. Note: If this manuscript was transferred from JNeurosci and a decision was made to accept the manuscript without peer review, a brief statement to this effect will instead be what is listed below.
Your manuscript was reviewed by two researchers and their comments are below. Please respond to each point denoted with an EDITORterm COMMENT in your summary of changes to the manuscript. Thank you.
Also, please move the Methods section to where it normally is in J Neurosci/eNeuro style articles.
Reviewerterm 1 The manuscript compare motor and cognitive performances of two age groupsterm·var (3 and 9 months) of C57term, FVBterm and C57term/FVBterm hybridterm·def mouse strainsterm. The hybridsterm·def tend to outperform both C57term and FVBterm strainsterm for several parameters in 9-month old mice and are therefore well indicated experimental models for neurobehavioral research with geneticterm tools. In addition, the authors demonstrate that optogenetic photo-stimulation experiments using optoelectronic probes can successfully be carried in C57term/FVBterm hybridterm·def strainsterm expressing ChR2.
Overall the data are clearly presented and the manuscript well written. My main critic is that the manuscript is somewhat misleading regarding the nature of the differences between species. The major claim is that hybridsterm·def outperform both progenitor strainsterm in several learning and motor tasks, and, in addition, show less aging-related deficits. The overall impression from title, abstract and main text is that hybridsterm·def outperform progenitor strainsterm regardless of age. However, according to the data (supplementary figures 2 and 3), there is not much difference between hybridterm·def and C57term strainsterm for the 3-month old mice. The differences are mostly significant for 9-month old mice. Hence, hybridterm·def and C57term mice start at the same level during early adulthood (3-months) and then C57term mice show reduced properties as they degrade faster with age.
I think the manuscript should be clearer regarding that the effect of agingterm is explaining the better properties of hybridsterm·def at relatively old age (9-months) and that hybridsterm·def present no major advantage when young adult mice are used (except for their larger size).
EDITORterm COMMENT: Please revise the manuscript in response to these comments. Addressing these points is key for your resubmission.
The data of the 9-month old mice is presented before the data of the 3-month old mice (which is introduced as to characterize the effect of agingterm), which is a bit confusing. I suggest presenting the 3-month old mice data first (or altogether with the 9-month old mice data, like in the supplementary figures).
EDITORterm COMMENT: Please reorganize the presentation of your data following the advice above minor comments: Introduction . “On the one hand, an organism as similar as possible to the object of interest is desired.”
maybe “an organism as similar as possible to human is most often desired" Introduction . “On the other hand, the least advanced organism for answering the research question is preferred. “ maybe adding an explanation such as “for ethical considerations” at the end FigS4: the “b” for Ab is missing EDITORterm COMMENT: Please revise to address the above three points.
Reviewerterm 2 The manuscript by SloinpersName et al., entitled “Hybridterm·def offspring of C57BL/6J mice exhibit improved properties for neurobehavioral research” produced and extensively tested the first-generation hybridterm·def offspring of FVBterm/NJ x C57BL/6J mouse strainsterm. Authors have comparatively tested Hybridterm·def (HYBterm) mice in the several behavioral paradigms, along with their electrophysiological properties and optogenetic assessments of their neurons. They report that HYBterm mice showed reduced anxiety-like behaviorterm, improved learning, and enhanced long-term spatial memoryterm. In addition, their age-related differences have been evaluated.
This study is well designed, nicely conducted and excellently written. I believe that this manuscript will be worth publishing in eNeuro following minor revision as suggested below.
1. Authors need to discuss that aging indexterm·def for HYBterm mice is significant thus what age of mouse suitable for anxietyterm studies. In addition, it would be interesting to know that how much anxietyterm resistance could persists in stressful situations in 9-month-old mice.
EDITORterm COMMENT: Please add comments to address these points in the revised Discussion section.
2. Authors used Kruskal Wallis test for several group's analysis. They need to explicitly mention in methods that why they used Non-parametric test.
EDITORterm COMMENT: Please clarify why non-parametric tests were used in the Methods section. Report results from exploratory data analysis to justify use of non-parametrics or state another reason.
3. The LED power used in optogenetic study was 11.07 μW, which seems very low. Is the unit used herein is correct?
EDITORterm COMMENT: Please confirm or revise the level of light used in the opto part of the study.
4. Since differential expression of viral vectors expressions observed in different neurons, authors need to show the pictures of their viral vector expression, for opto study. Also, briefly discuss the possibility of affected viral expressions, if any, in the HYBterm mouse brains.
EDITORterm COMMENT: Please add an image of a representative tissue as requested and add text to address these issues in the Methods or Discussion.
5. Give the information on statistical analysis software used in the study.
EDITORterm COMMENT: Please review terms used for statistical tests throughout the paper. It might not be clear to many readers what you mean by, e.g., U and G tests.